Biological control

ABSTRACT

The invention relates to a non-human multicellular organism carrying a dominant lethal genetic system, the lethal effect of which is conditional, wherein the lethal effect of the lethal system occurs in the natural environment of the organism.

[0001] The present invention relates to a method for controlling thepopulation of an organism.

BACKGROUND OF THE INVENTION

[0002] Methods of biological control are known for insects and plants.One method currently employed for the control of insect populations istermed the “sterile insect technique” (SIT), also known as the “sterileinsect release method” (SIRM). In this method, sterile males arereleased into the environment, wherein they compete with the wild-type(fertile) males for mates. Females which mate with sterile males produceno offspring, and the release of large numbers of sterile males,therefore, leads to a decrease in the size of the next generation. Inthis way the size of the wild population is controlled.

[0003] SIT requires some mechanism for insect sterilisation. Inaddition, SIT commonly also employs separation of males from females,with the release of only one sex. This is desirable in the case of anagricultural pest, such as the medfly, where the female damages fruit,even if the female is sterile. Similarly, only the female mosquito biteshumans. As such, release of the female insect is preferably avoided inthese cases.

[0004] Current techniques to achieve both sterilisation and separationof the sexes all have drawbacks. In some cases it is possible toseparate males and females by criteria such as pupal mass or time ofeclosion, but these methods are unlikely reliably to yield a trulysingle-sex population. Separation of males and females often involvesthe use of mutant strains, which have been mutagenised to induce avisible or otherwise selectable difference between the sexes, but suchmutagenesis can reduce the fitness of the resultant stock with respectto the wild type, which is undesirable.

[0005] Fitness may be further reduced in the sterilisation procedure, inwhich insects are given a sterilising dose of radiation (X rays or gammarays), or are chemically sterilised. Frequently, the doses of chemicalsor the dose of radiation required to induce sterilisation are verysimilar to that which is lethal for the organisms As such, sterileorganisms are frequently impaired in their ability to mate. Furthermore,both chemical and irradiation methods utilise technologies which are notspecific to the target organism, with consequent potential danger toworkers. Both methods produce an environmental hazard, as theirradiation source or chemicals will need to be disposed of In addition,there are inherent dangers and additional labour costs in the use of anirradiation source such as a strontium source.

[0006] Fryxell and Miller (Journal of Economic Entomology, Vol 88, No 5,pages 1221-1232) disclose an alternative strategy for insect control,using Drosophila containing a dominant conditional lethal gene which isexpressed under appropriate cold conditions in the wild. However, thismethod can be ineffective due to varying field conditions, where theenvironment does not provide suitably cold conditions. Moreover,organisms that live in a range of temperature habitats may not becontrolled under all conditions.

[0007] Asburner et al., (Insect Molecular Biology, 1998, 7(3), 201-213)disclose methods of transformation of insect species with foreign DNA,to produce transgenic species.

[0008] DeVault et al. (Biotechnology, Vol 14, January 1996, page 46-49)disclose a two-stage process which is a modification of the SITprocedure. Insects are initially separated by expression of a stablyinserted female specific promoter linked to a lethal gene, which isexpressed to kill females and to produce just one sex. The remainingmales can then be sterilised by irradiation or chemical treatment andreleased into the environment. However, this method suffers from thedrawback referred to above, in that released flies have reduced fitnessdue to the sterilisation treatment. Alternatively, the DeVault articlediscloses use of this genetic sexing step in combination with a secondgenetic system which may serve to sterilise or retard the hardiness ofthe natural population.

[0009] There is still a need in the art for a method of biologicalcontrol which avoids the problems with the above methods.

[0010] The present invention sets out to overcome such problems.

SUMMARY OF THE INVENTION

[0011] In a first aspect, the invention relates to a non-humanmulticellular organism carrying a dominant lethal genetic system thelethal effect of which is conditional, wherein the lethal effect of thelethal system occurs in the natural environment of the organism.

[0012] In a related aspect, the invention relates to an organism viablein a laboratory under controlled conditions. Controlled conditions areconditions that do not occur in the natural environment of the organism.As such, the conditions are typically artificial. Removal of thecontrolled conditions permits expression of the lethal genetic system.The organism may be autocidal, in that it will be killed after releaseinto the environment. Suitably the organism can transmit a lethalelement to at least some of its offspring, such that at least some ofthese offspring are also killed.

[0013] The organism of the invention can be used in population controlto pass on the lethal genetic system through mating, and also to blockpotentially productive mating of wild type organisms. Distribution ofthe organism of the present invention into the environment thusinitiates a biological control system. The organism of the presentinvention does not need to be sterilised, thus avoiding problems withsterilisation through irradiation and loss of genetic fitness.

[0014] In a further aspect, the invention provides a method ofbiological control, comprising:

[0015] i breeding a stock of male and female organisms under permissiveconditions, allowing the survival of males and females, to give a dualsex biological control agent;

[0016] ii releasing the dual sex biological control agent into theenvironment at a locus for biological control, and

[0017] iii achieving biological control through expression of thegenetic system in offspring resulting from interbreeding of theindividuals of the biological control agent with individuals of theopposite sex of the wild population.

[0018] Preferably there is no specific sterilisation step for releasedorganisms.

[0019] In addition, we have now discovered a new method for biologicalcontrol, applicable to organisms capable of sexual reproduction, whereinonly one lethal genetic system is required, the expression of which isused in both sex separation and biological control. In this case thelethal genetic system is made to be sex-specific. The lethal geneticsystem is preferably a conditional dominant sex-specific lethal geneticsystem, which is expressed in the restrictive conditions of the naturalenvironment of an organism. However, the expression of the lethalgenetic system may be controlled under permissive conditions in alaboratory, factory or other regulated system, for example, to allowgrowth of a normal populations, e.g. insect stock with both sexes. Priorto release of the factory or laboratory stock into the environment theconditions can be manipulated to ensure only single sex populations ofthe organism are distributed into the environment. No additionalirradiation of the organism is required and the arrangement removes anyrequirement for use of two separate genetic systems (i.e. those employedby De Vault et al, for sexing and, for example, sterilisation). Only onegenetic system needs to be constructed and inserted into the organism,which renders the methodology easier and quicker.

[0020] Thus, in a anther embodiment of the invention, the multicellularorganism carries a dominant sex-specific lethal genetic system which isconditional, and does not have a dominant sex-specific lethal geneticsystem which is unconditional and is expressed in every individual.

[0021] Specifically, under permissive conditions, the lethal geneticsystem in the organisms of this invention is not expressed, and a stockof organisms can be bred. Imposition of restrictive conditions thenallows one sex (for example, females) to be killed The remaining sex(males) can be released to the environment, and the genetic system ispassed on to at least some offspring resulting from any sexualreproduction between said males and a wild-type organism of the samespecies. The conditional dominant lethal genetic system is selected suchthat expression of the lethal system occurs in the natural environment.As a result, for a female specific lethal genetic system, all femaleswhich result from the mating are then killed or rendered non viable dueto the action of the genetic system, while the males survive to pass onthe system to the next generation in a proportion of cases. In this way,biological control is achieved.

[0022] If desired, the stock of organisms grown under permissiveconditions can be released into the environment, without imposing therestrictive conditions to kill off one sex before release. Thisvariation permits the possibilities of using a timing mechanism, e.g.life cycle stage, in creating a biological control agent. That is, theimposition of the restrictive condition is programmed by an event otherthan, for example, a pre-determined change in factory/laboratoryconditions prior to release into the environment For example, release ofa normal population of larvae creates a useful time-scatter or delayedrelease agent. By this is meant that individual larvae may proceed tomaturity at different rates and therefore release of the single sexgenetically engineered population could occur over a period of time andhence create a maximum probability of interaction with sexually activewild populations over that period This aspect may have advantages over asingle time point release of a single sex population of the geneticallyengineered adults. There arc other advantages, notably that the last(biggest) generation does not have to be reared in the factory,laboratory or other regulated environment, so saving space and food andthereby giving a more economic process. Moreover, the released larvaewill compete with the larvae of the wild population, increasingmortality through density-dependent mechanisms. By way of illustration,this variation might be useful with mosquitoes, where the larvae areharmless to humans, but not with medfly or codling moth, where thelarvae Therefore, in a further aspect the present invention provides amethod of biological control for an organism, the organism havingdiscrete sexual entities, the method comprising the steps of:

[0023] 1 production of a stock of genetically engineered organism;

[0024] 2 release of the genetically engineered organism into theenvironment either as

[0025] a) a normal population (i.e. containing both sexes) at a certainstage of the life cycle of the organism, e.g. larvae, in the knowledgethat females will die and only males will mature into adults, or

[0026] b) a single sex population, i.e. after the sex specific dominantlethal effect has been expressed prior to release.

[0027] The invention relies on expression of a conditional dominantlethal genetic system capable of sex specific lethality, in order toeliminate one sexual entity. The conditional expression of the lethalgene is such that the lethal effect occurs in the natural environment ofthe organism to cause the biological control.

[0028] In a yet further aspect of the invention, the inventionaccordingly involves a third step;

[0029] 3 allowing biological control to occur.

[0030] The invention further provides a method of biological control,comprising:

[0031] breeding a stock of males and female organisms under permissiveconditions, allowing the survival of males and females, to give a dualsex biological control agent;

[0032] optionally before the next step imposing or permittingrestrictive conditions to cause death of individuals of one sex andthereby providing a single sex biological control agent comprisingindividuals of the other sex carrying the conditional dominant lethalgenetic system;

[0033] releasing the dual sex or single sex biological control agentinto the environment at a locus for biological control, and

[0034] achieving biological control through expression of the geneticsystem in offspring resulting from interbreeding of the individuals ofthe biological control agent with individuals of the opposite sex of thewild population.

[0035] The invention also relates to organisms comprising a conditionaldominant lethal genetic system for use in a combined method of sexseparation and biological control as herein defined.

[0036] The invention further provides a multi-phase lethal system havinglethality at more than one life cycle stage. Specifically, the inventionprovides an organism or single sex population for use in biologicalcontrol, wherein the organism or single sex population produces noviable progeny when mated with the wild-type opposite sex underrestrictive conditions, e.g. in the natural environment. For example,the invention provides a male population which produces no viable maleor female progeny. This contrasts with the situation in which a maleonly population produces no female progeny but viable male progeny.

[0037] The invention further provides a method for the sex-separation oforganisms, wherein the expression of a sex specific dominant conditionallethal system is used to kill one sex to leave either an essentiallypure male or female population, or a population in which organismscomprise either male or female tissues, or a population in whichorganisms are unable to produce functional male gametes or femalegametes (or both) which they would have been able to produce but forexpression of the lethal genetic system.

[0038] The invention further provides a method of biological control inwhich the growth of a stock of organisms under permissive conditions,once initiated, is self-sustaining and requires no additional pool oforganisms for its maintenance.

[0039] The invention further provides a method of biological control inwhich the expression of the lethal genetic system occurs in the absenceof a substance which is absent from the natural environment of theorganism, thus ensuring effective biological control when the organismis released.

[0040] The invention further provides a vector for use in transformationof an organism to produce an organism according to the presentinvention, suitable for use in a biological control scheme.

GENERAL DESCRIPTION OF THE INVENTION

[0041] The general features of the invention are first outlined in broadterms for ease of understanding, before being specifically detailed. Theinvention is discussed herein with respect to both organisms for use ina method of biological control and methods of biological control.Reference to an organism thus generally is taken to include a method ofbiological control employing that organism, and vice versa.

[0042] The non-human organism of the present invention is suitably arecombinant organism, into which the dominant lethal genetic system hasbeen transformed. The organism is also at least capable sexualreproduction or attempting sexual reproduction, such that the dominantlethal genetic system can be passed into the naturally occurringpopulation of that organism, or the organism can compete with wild typeorganisms in mating.

[0043] The lethal genetic system is suitably comprised of a lethal geneand controlling and/or regulatory elements. However, in one embodiment,the lethal system may be comprised simply of a lethal gene, sufficientto produce the lethal effect.

[0044] The dominant genetic system suitably includes a dominant genewhose effect is phenotypically expressed in the heterozygous state. Thisdominant effect ensures that, if an organism only receives one copy ofthe lethal genetic system, then the lethal effect of that system willnevertheless be exerted in the host in the natural environment of theorganism.

[0045] The lethal genetic system may be sex-specific or non-sexspecific, the former being generally preferred. In the case of asex-specific lethal system it is possible to carry out a geneticsex-selection before release of organisms for biological control.

[0046] When a single sex biological control agent is desired, separationof the sexual entities is normally achieved in the method by removal ofpermissive conditions while a stock of an organism is grown up,resulting in the sex specific lethal effect of the genetic system beingmanifested. A single sex population remaining may then be isolated.

[0047] We prefer that the lethal effect is female specific. However, amale specific lethal effect may be required in certain situations. Withreference to plants, the sexual entities need not be discrete organisms,but parts of the same organism. The present invention may thus also beapplied to plants, wherein one sexual entity of a plant is killed. Witha single sex biological control agent, the conditional dominant lethalgenetic system is permitted to be expressed during growth cycles beforerelease, and the plant then distributed. Alternatively, no suchpermissive expression might be needed before release, for instance inthe case of seed distribution with the lethal effects only manifestingonce the plant reaches a certain further stage in its life cycle in theenvironment.

[0048] With respect to insects and other animals, distributing theorganism typically occurs by release of the organism into theenvironment. With plants, distributing typically occurs by planting ofmature plants, seedlings or seeds, or any suitable form of the organismin the environment.

[0049] The conditional effect of the dominant lethal genetic system isseen except under defined permissive conditions. In the presentinvention the restrictive conditions occur in the natural environment ofthe organism, and are those conditions which allow the lethal effect ofthe lethal system to be expressed. The permissive conditions which allowthe survival of the organism are only present when adopting permissiveconditions in the regulated growing environment.

[0050] Preferably expression of the dominant lethal genetic system isconditional upon the presence of a substance or condition not found inthe natural environment, such as an artificial or synthetic compound,suitably an antibiotic, antibiotic analogue or derivative. Such anartificial substance or condition is suitably always absent from thenatural environment, that is, it is never or only rarely present in thenatural environment in sufficient abundance or concentration toinactivate or functionally repress the lethal genetic system. Preferablyabsence of the substance or condition results in expression of thelethal effect of the lethal system.

[0051] The natural environment of the organism is generally theenvironment in which the population to be controlled is located, or maysurvive. Additionally, the natural environment is also an environmentwhich provides the necessary restrictive conditions. The universalnature of the invention allows universal application of the methods usedin the invention, and the natural environment may thus be any worldenvironment in which biological control is needed, without restriction.

DETAILED DESCRIPTION OF THE INVENTION

[0052] The lethal genetic system of the present invention may be anygenetic element or combination of elements which is capable of producinga lethal effect. We prefer that the lethal genetic system comprises aDNA sequence encoding a potentially lethal gene product (a lethal gene)and controlling elements such as promoters, enhancers or trans-activatorcomponents. The elements which regulate the gene may be located on thesame chromosome as the lethal gene, which is preferred, or on adifferent chromosome. We particularly prefer that the lethal system is alethal gene the expression of which is under the control of arepressible transactivator protein. In an alternative embodiment thelethal system may simply be the lethal gene alone, or in combinationwith its native promoter.

[0053] Preferably the organism of the present invention has only onelethal genetic system, the system wing conditional on environmentalfactors. More preferably the system has only one conditional lethalgene. The use of a simple genetic system minimises the chance of geneticcomplication when producing or carrying out the invention. Typically theorganism contains no transgenes or other non-natural gene or DNAarrangements, other than that of the lethal genetic system of theinvention.

[0054] The lethal effect of the lethal system may affect the wholeorganism, or be targeted to specific tissues within an organism. Forexample, in plants the lethal effect may be targeted to only a part ofthe host plant, such as one of the sexual organs of the plant. As such,in the present invention, a reduction in the wild type population sizeis achieved without the use of applied sterilisation by externallyapplied agents such as irradiation or chemicals, but through the use oftargeted lethality based on zygotic lethality, or male or female ortotal sterility.

[0055] In particular, in plants, we prefer that precursors of the maleand/or female gamete-producing tissues or critical parts thereof withinthe plant are targeted by the lethal effect, such that these tissues diewhen the plant is grown in the natural environment. In this way, theplant will produce no pollen or seed, or neither pollen nor seed, unlessgrown under permissive conditions. Given general environmental concernsover genetically modified crops, this invention is therefore especiallyuseful when applied to plants which are transgenic at another locus. Thetransgenic plant will then release no pollen or seed, and cannot crosspollinate other species or otherwise spread into the environment. Thisis of especial benefit where the plant has wind-blown pollen. In thisway, the transgenic plant is contained, and can be grown in fieldstudies for testing prior to commercialisation without risk to theenvironment.

[0056] The invention thus relates to a method for the field testing oftansgenic crops, comprising the Step of growing a transgenic plantcomprising the conditional lethal dominant system of the invention underpermissive conditions, and then distributing the plant into theenvironment where it is exposed to restrictive conditions. A field testis generally any test carried out on a transgenic plant to asses itscharacteristics, such as its commercial suitability as a crop orfoodstuff, for example. The invention also extends to plants having theconditional lethal dominant system of the invention in combination withone or more transgenes.

[0057] The lethal effect may also be targeted to a specific life cyclestage of the organism. Where life cycle specificity is sought, we preferthat the lethality of the invention is embryo specific lethality. Thelethal phase suitably ends before the developmental stage at which theorganisms are released, or they may lose fitness or die followingrelease. In the case of insects, embryonic lethality ensures that nolarvae emerge to damage crops or animals. Whilst this is less importantin the case of disease vectors such as mosquitoes, where only the adultstages transmit the disease, it is important in the case of many croppests where it is the larvae that cause economic damage. Embryo-specificlethality allows the last and biggest mass-reared generation to bereared on food lacking the repressor, reducing costs. Embryo-specificlethality can also be combined with later sex-specific lethality, e.g.female-specific lethality. In this case we demonstrate that this allowsthe construction of a strain in which both sex-separation and“sterilisation” are automatic consequences of the withdrawal ofpermissive conditions from the last generation prior to release.

[0058] Also preferred, in certain circumstances, is late-actinglethality, which takes advantage of the feature of density-dependentnegative selection, in which the chances of an individual surviving toreproduce is negatively dependent of the total number on individuals inthe population of which it is a part. The mechanism for this istypically competition between individuals for limited resources, such asfood. By way of example, in the case of mosquitoes, this competitionmight act on larvae competing for food. If the lethal phase is laterthan this larval competition stage, then the individuals (e.g. femalelarvae) who will be killed by the lethal system will nonetheless competefor resources during their larval stage and so indirectly reduce thenumbers of their conspecifics, even those that do not carry the lethalsystem at all. Therefore, preferred is a lethal system which is lethalat a life cycle stage which allows competition between organisms of theinvention and wild type organisms to occur.

[0059] Preferably the lethal expression is such that individuals diebefore they cause the damage which it is intended to prevent. By way ofexample, in the case of mosquitoes it is desirable to reduce diseasetransmission. The earliest that a female mosquito can transmit diseaseis the second blood meal (having picked up the parasite/virus in thefirst blood meal and so become infectious). Therefore, the mosquito canbe killed as late as shortly after the first blood meal. In addition,mosquito feeding is also undesirable, and preferably killing is effectedshortly before or just after the first blood meal.

[0060] The lethal gene of the lethal genetic system may be any geneticelement which is capable of causing the death of, or leading to thefatality of, the host. In particular, the term covers gene fragmentscapable of exerting a lethal effect, and is not limited to full lengthgenes. Any element capable of exerting a lethal effect which may beconditionally controlled is covered by this term.

[0061] The choice of dominant lethal gene is not critical to theinvention. There is a wide range of suitable gene products, with varyingtoxicities. For example, dominant mutant forms of cell-signalling orcell-cycle genes are appropriate for use in the present invention.Constructs which result in overexpression of such genes may also belethal. Similarly constructs which result in inadequate expression ofany essential gene would also be lethal. This might be achieved byexpression of an inhibitory sequence, for example antisense RNA, senseRNA (acting by gene silencing), double stranded RNA (“inhibitory RNA” orRNAI) or other inhibitory RNA molecule. Overexpression of proteininhibitors of essential functions could also perform this lethalfunction. Other suitable targets for engineering constructs includegenes which disrupt metabolism or regulation of the cell to a fatalextent, such as disruption or overexpression of extracellular signallingfactors such as functional homologues of Wnt, Shh or TGFβ. Preferredlethal genes are those described in the Examples herein, the hid gene[see Heinrich and Scott, P.N.A.S Jul. 18 2000, volume 97, 15,8229-8232], and the Nipp1Dm gene, a Drosophila homologue of mammalianNIPP1 (see Example 7). Other possibilities for lethal genes includesex-determination genes which may act to tnansform the sex of theorganism. In this case, transformation of females to sterile males wouldalso enable biological control to be achieved, and the lethal gene islethal to the population as such and not specifically to the organism.Where highly toxic gene products are used, such as diphtheria toxin andricin A, we prefer that the genes are only expressed at levelssufficient to kill the organism, but with minimum environmental impact.

[0062] A preferred lethal gene for use in the invention has a thresholdof toxicity—below a certain level it is harmless while above it islethal. Additionally, to reduce the possibility of resistance, thelethal gene preferably has multiple essential targets. Nipp1Dm generallyfulfils these criteria It encodes a highly conserved protein present inall cells at a significant level. Modest over-expression is thereforeunlikely to have any adverse consequences. It is a potent inhibitor ofthree essential genes in Drosophila, each of which have highlypleiotropic effects. Accordingly, because of the high level ofconservation of this protein between C. elegans, D. melanogaster andmammals, Nipp1Dm is a preferred lethal gene for use in the presentinvention.

[0063] The conditional nature of the lethal system allows recombinantorganisms to be bred under conditions permissive for organism survival,for example in a factory or laboratory, and then released into thenatural environment The lethal effect of the lethal system is controlledsuch that the released organisms are able to breed, and sexualreproduction allows the lethal system to be passed into the wild typepopulation, killing all or a defined group of these organisms. We preferthat the lethal effect results in killing of greater than 90% of thetarget class of the progeny of matings between released organisms andthe wild population. The target class may be, for example, females, i.e.50% of the progeny. More preferably the lethal effect results in killingof greater than 95% of the target class, still more preferably 99% andmost preferably 100% of the target organisms in the environment. Theconditional nature of the lethal system may be conditional on anysuitable factor, such as temperature, diurnal cycle (with light durationand/or intensity being factors) or pheromones, for example. In thiscase, the recombinant stock could be reared at the permissivetemperature, and released into an environment having a restrictivetemperature. Suitably the lethal effect occurs at a temperature which isat least 5° C., more preferably 10° C., more preferably 20° C., withinthe extremes of the temperature range known to occur in the environmentof the organism across the world, such that there is always expressionof the lethal effect in the environment.

[0064] Preferably the lethal effect of the lethal system is inherentlyinsensitive to temperature variations or fluctuations which occur in thenatural environment of the organism.

[0065] Where the expression of the lethal system is not conditional ontemperature but is temperature sensitive to any extent, we prefer thatgreater than 90% of the organisms are killed in the natural environment,more preferably at least 95%, preferably at least 98%, preferably atleast 99% or more.

[0066] The lethal genetic systems of the present invention are generallynot susceptible to temperature to any significant extent, so that forexample, the difference in lethal effect at 1° C. and 29° C. is lessthan 5%, preferably less than 1%. The preferred lethal genetic systemsof the invention are suitably functional across a broad temperaturerange, such as may occur naturally within the environment where theorganism is found. Examples of typical temperature ranges are 0° C. to50° C., more usually 10° C. to 45° C., such as 15° C., 20° C. or 25° C.40° C. Preferably the lethal effect is exhibited in at least 95% oforganisms across this whole temperature range, in that 95% of organismsare killed at any given temperature in the range, more preferably 98%,99% or even more. More generally, the highest survival rate at anytemperature is preferably less than 10%, suitably 5%, 2%, 1% or less.

[0067] The lethal effect of the lethal system is preferably expressed inthe natural environment when the organism is distributed into itsnatural environment or any naturally occurring environment,irrespective-of the natural conditions which can occur or which prevailin that environment.

[0068] We prefer that the lethal effect of the lethal system isconditional upon a dietary additive, such as a food or water additive,which is not a normal food component for the target species. This allowsthe recombinant stock to be grown on food or water containing theadditive, which prevents the lethal effect. On release into the wild,the organism has no exposure to the additive, and the lethal effect ofthe lethal system is expressed in the progeny of a mating with therecombinant organism of the invention. It may also be expressed in theparent organism under certain circumstances, although the releasedorganism must survive long enough to mate.

[0069] Preferred factors on which the expression of the lethal systemcan be made conditional include antibiotics such as tetracycline andnon-antibiotic tetracycline analogues and derivatives thereof, whichfunction with the preferred tetracycline repressible system of thepresent invention. Non-antibiotic compounds are especially preferred toavoid potential problems with antibiotic accumulation in theenvironment. Suitable analogues include epioxytetracycline andanhydrotetracycline, although other suitable analogues may also beemployed, as appropriate.

[0070] Where the lethal effect is conditional upon a dietary additive,it may be that the progeny will survive without themselves ingesting orabsorbing the dietary additive. For example, the progeny might retainsufficient of the additive from their parents or from an earlier lifecycle stage without feeding, or at the least the additive may be slowlylost from the progeny. This effect might pass through one or moregenerations before the lethal effect is fully expressed underrestrictive conditions.

[0071] We prefer that the recombinant multicellular organism of thepresent invention contains a dominant lethal system the lethal effect ofwhich is conditionally suppressible, In this way, the lethal effect issuppressed under controlled conditions, but not suppressed in thenatural environment of the organism. However, there may be other ways toattain conditional expression (for example, conditional activation), anyof which may be used in the present invention.

[0072] We particularly prefer that the repressible expression system isa tetracycline repressible system in which tetracycline, or an analogueor derivative thereof, is used to inhibit expression of the lethalsystems One suitable system is described in detail in the examplesherein, in insects. This tetracycline system has also been shown to workin plants (see Zuo and Chua, 2000, Curr. Opin. Biotech. 11:146 andreferences therein).

[0073] The repressible lac repressor system is less preferred, as theinducer (IPTG) is less diffusible and more toxic than tetracycline.

[0074] By way of contrast, an inducible system may be based upon theconstitutive expression of a toxin and inducible expression of arepressor of the toxin. One such example described in Zuo and Chua(supra) in relation to plants is based on expression of a chimerictranscription factor which is normally inactive (sequestered by bindingto Hsp90). In the presence of the inducer (a steroid hormone oranalogue, e.g. dexamethasone), the transcription factor is released fromHsp90 and can drive gene expression.

[0075] The components of this system are

[0076] i Promoter—toxin ORF

[0077] ii Promoter-transcription factor ORF

[0078] iii Transcription factor-responsive promoter—antidote ORF

[0079] Suitably, the tapetum-specific A9 promoter may be used. Thetapetum is a tissue required for production of functional pollen Thesystem is then ‘off’ in all tissues except the tapetum. In the tapetum,the toxin and the transcription factor are both expressed. In presenceof the inducer (here dexamethasone), the antidote is also expressed. Soplants treated with dexamethasone arc normal, but those not treated withdexamethasone produce no pollen.

[0080] Suitably barnase and barstar (Hartley, R W, 1988, J. Mol. Biol.202:913, Hartley, R W, T.I.B.S. 14: 450-454, 1989) may be used as toxinand antidote, respectively. However, while Barnase and Barstar aresuitable examples of a toxin/repressor pair, the invention is not solimited, and a suitable repressor could act at a transcriptional (orother) level, and the toxin itself does not have to be a protein.

[0081] It is the lethal effect of the lethal system which isconditional, and not solely the expression of the lethal gene.Therefore, the invention includes the possibility of conditional controlboth at the level of lethal gene expression, and by control of theactivity of the lethal gene product. As such, the invention includes thecase in which the lethal gene product is being produced but the effectof which is masked in some way.

[0082] We prefer that the method of the invention uses only organismswith a single conditional dominant lethal genetic system. In addition,we prefer that this system is the only recombinant element present inthe organism. We particularly prefer that the organism contains only onetype of lethal gene, but it is possible to envisage multiple lethalgenes under the same regulatory control, giving the integrated geneticconstruct concept but a more efficient lethality of the system. Thissingle lethal gene may be under the control of just one promoter in thegenetic system, or more than one promoter.

[0083] The organism of the invention is preferably recombinant, whichrefers generally to any organism whose genetic material has been alteredby genetic manipulation. We prefer that the organism is modified byinsertion of a gene, gene fragment or genetic element (such as apromoter or enhancer) from another species, to produce a transgenicorganism. The transgenic component is generally the lethal system whichproduces a conditional lethal effect. However, a conditional lethaleffect may also be generated using genetic components derived from thesame (host) species. For example, a promoter derived from a differentgene in the same species; when placed in front of a gene which is onlynormally expressed at low levels, may result in a lethal effect. Therecombinant organism is thus either a transgenic organism or one inwhich the host genetic material has been modified to produce a lethalsystem.

[0084] The multicellular organism may be any organism, such as a plantor animal. Indeed, the invention is generally only limited to thoseorganisms having a sexual component in their life cycle, which enablesthe lethal system so be transferred from one organism to another. Forexample, the invention is also applicable to fish, such as the sealamprey, against which sterile male release techniques have beenemployed. We particularly prefer that the multicellular organism of theinvention is an insect, with insect pests being particularly preferredAn insect pest may be either a direct or an indirect pest Direct pestsare those insects which cause damage at one or more stage of their lifecycle by, for example, eating crops or damaging animals The New Worldscrew-worm fly Cochliomyia hominivorax, for example, is a direct pest ofcattle. Indirect pests are those insects which are vectors of humandiseases, such as mosquitoes which carry malaria Indirect pests oforganisms other than humans, such as livestock or plants are also known.

[0085] Preferred insect targets for the present invention include Crop(arable and forestry) pests animal pests and disease vectors. Examplesof specific organisms which potentially may be used in the presentinvention include, but are not limited to: Australian sheep blowfly(Lucilia cuprina, Asian tiger mosquito (Aedes albopictus); Japanesebeetle (Popilla japonica), White-fringed beetle (Graphognatus spp.),Citrus blackfly (Aleurocanthus woglumi), Oriental fruit fly (Dacusdorsalis), Olive fruit fly (Dacus oleae), tropical fruit fly (Dacuscucurbitae, Dacus zonatus), Mediterranean fruit fly (Ceratitiscapitata), Natal fruit fly (Ceratitis rosa), Cherry fruit fly(Rhagoletis cerast), Queensland fruit fly (Bactrocera tryoni), Caribbeanfruit fly (Anastrepha suspensa), imported fire ants (Solenopis richteri,Solenopis invicta), Gypsy moth (Lymantria dispar), Codling moth (Cydiapomonella), Brown tail moth (Euproctis chrysorrhoea), yellow fevermosquito (Aedes aegypti), malaria mosquitoes (Anopheles gambiae,Anopheles stephansi), New world screwworm (Cochliomyia hominivorax), OldWorld Screwworm (Chrysomya bezziana), Tsetse fly (Glossina spp), Bollweevil (Anthonomous grandis), Damsel fly (Enallagma hageni), Dragonfly(Libellula luctuosa), and rice stem borer (Tryporyza incertulas).Reviews discussing the suitability of many of the above are: C. Boake etal., (1996) Annu. Rev. Entomol. 41: 211-219, J. Meyers et al., (1998)Annu. Rev. Entomol. 43: 471491, C. Calkins et al., (1994) Fruit fliesand the sterile insect technique. CRC Press. ISBN 0849348544, E. Krafsuret al., (1997) Annu. Rev. Entomol. 42: 503-523 and R. de Shazo et. al.,(1994) J. Allergy Clin. Immunol. 93(5): 847-850. It will be understoodthat the present invention is generally applicable to all multicellularorganisms capable of sexual reproduction, such as plants and animals.

[0086] For all animals, the transgenic stock is released into theenvironment at appropriate sites and times. For plants, where the adultsare not mobile, the procedure is slightly different. Either the gametesthemselves are released, e.g. as pollen, or plants are dispersed, e.g.at field margins, to pollinate wild weeds and so reduce theirreproductive potential. The present invention is of particular use inthe control of those weeds, such as rye grass, which are not wellcontrolled by current herbicides, or against weed types which havedeveloped herbicide tolerance.

[0087] Not all of the terms which are used to describe, for example,plants are applicable to animals or vice versa. However, the principlesof the invention as laid out in relation to one species may readily beapplied to other species by a person skilled in the art For example,where the terms ‘female’ and ‘male’ are used in relation to insects,these may also refer to plants having only viable female or male tissuesrespectively, where appropriate. The term ‘sex separation’ also mayrefer to plants which have been separated on the basis of their viablesex tissues from other plants.

[0088] The invention is preferably such that expression of the lethalgenetic system will always occur in the environment in which theorganism is released for biological control, and is unaffected bynatural variation in environmental factors. In this way, biologicalcontrol is always achievable using the present invention, irrespectiveof the site of release, time of release, or any other environmentalconditions. Where the factor controlling conditional expression isartificial, then it is immediately clear such a factor cannot, bydefinition occur in the natural environment. The present invention isessentially pandemic, in the sense that it may be universally appliedover the whole of a country or the world environment.

[0089] Essentially any natural environment itself provides therestrictive conditions for the organism, resulting in the biologicalcontrol. As such the restrictive conditions are guaranteed to occur uponorganism release, and there is no concern that local environmentalconditions will affect the action of the lethal system. Preferably thenatural environment of the organism provides the absence of acontrolling factor or condition, which then results in expression of thelethal genetic system in the environment.

[0090] The multicellular organism of the present invention preferablyhas a lethal system homozygous at one or more loci. In the situationwhere there is one homozygous copy of the lethal system, then at leastone copy of the system will be passed to any offspring during sexualreproduction. Therefore, the dominant lethal effect will be exerted,except in permissive conditions. The present invention may be carriedout using a heterozygote for the dominant lethal system. However, inthis case, not all the offspring will have a copy of the lethal system,and the effect on the population is reduce.

[0091] It is preferred that all the elements of the genetic system arepresent on the same chromosome, in close proximity. In this way, it islikely that all elements of the lethal system are passed on tosubsequent generations. However, the lethal system can also functionwhen controlling elements are present at different genetic loci to thelethal gene, if controlling effects of these elements are exerted intrans, for example. In that event, the genetic system is still effectiveif the controlling and lethal elements are also homozygous, and at leastone copy of each is transferred to the offspring.

[0092] In one aspect the invention relates to a non sex-specific system,in which both males and females are killed by the lethal genetic system.Such an approach is preferred in certain organisms. In such a case, oneadvantage of the invention lies in the avoidance of sterilisation byirradiation. By way of example, mixed sex releases are preferred in pinkbollworm (a lepidopteran pest of cotton), but irradiated moths areestimated to suffer at least a 10 fold reduction in effectiveness as aconsequence of the irradiation due to loss of vigour and reduced lifespan. Similar advantages are predicted in other organisms. In medfly,irradiated males are about 50% less effective than the non-irradiatedequivalent in competitive mating tests and they live 3-5 days instead ofthe non-irradiated 10-15. This gives a composite 410 fold potentialperformance improvement by avoiding irradiation.

[0093] The method of the invention alternatively uses a sex-specificlethal system to achieve sex separation before or after release oforganisms into the environment. In a prefer embodiment, themulticellular organism is an insect containing a homozygous dominantlethal system, the lethal effect of which is lethal only to females. Inthis embodiment males released into the natural environment will not bekilled After mating with females, female offspring will contain at leastone copy of the dominant system and be killed. However, male offspring,50% of which contain the dominant system viable and may mate withfurther females. In this way, the dominant system may be transmitted tosubsequent generations, although without further artificialintroductions the system will eventually be lost from the gene pool.

[0094] In the case in which a male contains a lethal genetic system witha female specific lethal effect, then males released into theenvironment will not be killed. However, the lethal effect of the lethalsystem is still manifested in the natural environment—even if thiseffect is limited to females.

[0095] Sex-specific lethality may be achieved in a number of differentways. For example, it is possible to use a sex-specific lethal gene aspart of the lethal system, whose gene product is toxic only in one sex.This approach will allow killing of a single sex even if expression ofthe lethal gene of gene product is not sex specific. Candidates forfemale sex-specific lethal genes include genes from the sexdetermination pathway, for example normally active only in males andtoxic in females, or genes derived from sexual differentiation orgametogenesis systems.

[0096] Alternatively, expression of the lethal gene or gene product maybe controlled so that it is expressed or produced only in one sex (or inonly one gamete or sexual organ of a hermaphrodite). For example,sex-specific promoters or enhancers may be used, either in combinationwith sex-specific lethal genes or non-specific lethal genes.Sex-specific splicing provides another mode for sex-specific geneexpression. All possible combinations of non-specific lethal genes,sex-specific lethal genes, non-specific promoters and sex-specificpromoters are envisaged by the present invention. In addition, othersex-specific factors which control the lethal effect of the lethal geneare included in the present invention.

[0097] The present invention also includes a method of biologicalcontrol in which the lethal effect may be sex-specific at one stage ofthe life cycle, but be lethal to both sexes at another stage. Forexample, the lethal system may be female specific in an adult organism,but be lethal to both males and females in the larval stage. In such acase, one sex may be killed by expression of the lethal system in theadult form. When the organism then breeds in the wild, passing on thegenetic construct, then both males and females can be killed. Such aneffect can be achieved by a promoter which is sex specific at one lifecycle stage, but not at another, or by placing the lethal gene undercontrol of two different promoters, for example. Multiple lethal systemsmight also be employed.

[0098] For example, a lethal effect manifested at an embryonic or larvalstage will not affect adult organisms, if they are grown underpermissive conditions through this stage. As such, organisms may bedistributed into the environment after the lethal life cycle stage,allowing the lethal system to be passed into the wild-type populationthrough sexual reproduction. Other life cycle stages, such as the adultstage, may also be targeted by selection of genes or promoters expressedat specific life cycle stages, if appropriate.

[0099] We prefer that the multicellular organism of the presentinvention has a copy of the lethal genetic system at more than onelocus. Preferably, the lethal system is homozygous at more than onelocus.

[0100] Multiple copies of the lethal system are useful to enhance theeffect of the invention. For example, if the organism is homozygous atone locus for a female specific lethal system, any females that resultfrom mating of the organism with wild type females will be killed. Maleoffspring will survive, and carry one copy of the system. Only 50% ofthe next (second) generation of male offspring will carry the lethalsystem.

[0101] The approach will clearly be more effective if more than 50% ofthis next (second) generation of male offspring were to inherit thelethal genetic system. There are several ways of achieving this. Forexample, if the lethal genetic system is homozygous at more than one,not tightly linked locus, e.g. on more than one chromosome, then theproportion of these males carrying the lethal genetic system willincrease. Specifically, with the lethal genetic system homozygous at twounlinked loci, the first generation males will be heterozygous at bothloci, 75% of the second generation males will carry at least one copy ofthe lethal genetic system. Correspondingly, under restrictive conditionsall of the first generation and 75% of the second generation femaleswill die.

[0102] Another way of achieving this effect is to use a segregationdistortion/meiotic drive system. In the Drosophila SD system, the SDchromosome is preferentially inherited from males heterozygous for SDand a normal (+) SD-sensitive chromosome. SD/+males transmit SD-bearing,to the virtual exclusion of +-bearing, homologues; as many as 99% of thefunctional sperm may carry SD. Segregation distortion/meiotic drivesystems are known in a wide range of insect and non-insect species.

[0103] A third way of ensuring >50% inheritance of the lethal geneticsystem in the second generation is to link the lethal genetic system toinsecticide resistance and use the insecticide to eliminate some or allof the second (and subsequent) generation progeny which do not carry thelethal genetic system and hence do not carry the linked resistance gene.

[0104] The lethal system may be located on any chromosome, either anautosome or sex chromosome. In species where sex is determined by the Xor Y chromosome content and where elimination of the transgene from thegene pool is desired, then we prefer that the lethal system is locatedon the X chromosome. Consider the case in which the lethal system isspecific for females. A male organism (XY) having the lethal system onthe X chromosome mates in the wild with a female wild type organism(XX). The male offspring must derive their Y chromosome from therecombinant male and their X chromosome from their mother. These malesare viable and have no lethal gene. Female offspring must derive one Xchromosome from the recombinant male and, thus, contain the lethalgenetic system—they are killed. As such. the lethal system is eliminatedfrom the gene pool, which may be preferable if this element is atransgene.

[0105] The present technology also provides a method for the selectionof males or females per se, comprising producing a organism as describedherein containing a conditional dominant lethal system, wherein thelethal effect of the lethal system is sex-specific. Sex selection isachieved by allowing expression of the lethal effect of the lethalsystem, to eliminate one sex. The individual male or female populationmay then be used for any desired purpose, not being limited tobiological control.

[0106] The present invention also relates to a method of producing arecombinant multicellular organism for use in the present invention,wherein the organism is transformed with a vector or vectors containinga dominant lethal system, or a suitable sequence for site specificmutation.

[0107] The present invention further relates to a vector or vectorscomprising a dominant lethal system as described herein.

[0108] We prefer that all the required elements to control theexpression of the dominant lethal gene are present on a singletransformation construct (vector). In this way, only a singletransformation step, and single transformation marker, are required. Inaddition, use of a single transformation construct helps preventrecombination of separate elements of the lethal genetic system.Therefore, preferred is a single vector comprising any conditionaldominant lethal genetic system of the invention.

[0109] Further preferred are vectors comprising the conditional dominantlethal genetic system of the invention, wherein the components of thevector (such as the genes or regulatory elements, in particular thelethal gene) are genetically insulated from one another. Preferbly thereis no cis cross-talk between the different elements of the lethalgenetic system of the vector. Preferred are vectors in which thecomponents of the lethal genetic system are separated from one anotherby insulator sequences derived from vertebrate DNA which prevent suchcross-talk. Such insulators have been reported to work in Drosophila,for example [Namciu, S. J., et al., (1998) Mol. Cell. Biol. 18: 2382-91and Chung, JH., et al., (1993) Cell 74:505-514], and by extension arelikely to be effective in other insect species at least.

[0110] In a preferred embodiment, the vector of the invention comprisesa tetracycline repressible system. Preferably a lethal gene is locatedon the same DNA sequence or vector as this system, optionally with areporter gene. A suitable tetracycline based lethal system comprises twokey components, a lethal gene and a tTA gene which activates expressionof the lethal gene Tetracycline, or analogue thereof, then blocksactivation of the lethal gene by the tTA. In this case, we prefer thatenhancer-blocking insulators are used to isolate one component from thenext, namely the lethal gene from the tTa, the lethal gene from thereporter and the tTa from the reporter gene.

[0111] A particularly preferred vector in which the genetic elements areseparated and modular is presented in the Example 7 herein This vectorcomprises a dominant lethal tetracycline repressible. genetic system,wherein at least somne of the genetic components of the system areseparated by genetic insulator sequences. The lethal gene is the Nippgene from Drosophila

[0112] This modular vector may be adapted by replacing the BmA³ promoterwith any suitable promoter to allow the construct to be used in anyorganism of interest., As such the invention provides a modular templatevector as described herein, where the BmA³ promoter module may bereplaced by any promoter, for use in any suitable organism.

[0113] The invention also extends to variants of this specific modularvector, in which the functional elements have been replaced with otherelements which perform equivalent functions, such as other insulators orlethal genes, and to DNA encoding such variants.

[0114] The invention also relates to a method of constructing a vectorappropriate for imparting a dominant lethal genetic system to anorganism, comprising the steps of:

[0115] i providing at least one conditional lethal genetic system;

[0116] ii choosing a promoter appropriate for expression of the systemin the organism; and

[0117] iii ligating the promoter and conditional lethal genetic system,optionally with other components, to produce a functional vectorsuitable for transformation;

[0118] wherein transformation of the vector into the organism producesan organism for biological control according to the invention.

[0119] Preferably the lethal genetic system of the vector is modular inthat there are components which can be individually replaced byfunctionally equivalent genetic components, appropriate for the lethalsystem to function in an organism of interest. For example, such amodular vector allows the lethal gene or promoter sequences to bereplaced, for example, without the need to generate an entirely newvector. Suitably the individual genetic components may be separated byinsulator sequences and still function together to cause a lethaleffect. Preferably the vector comprises at least one insulator sequence,preferably two such sequences.

[0120] The invention also relates to vectors obtained and obtainable bythe above method

[0121] The present invention also extends to polynucleotide sequencesencoding a conditional dominant lethal genetic system according to thepresent invention, preferably being a DNA sequence. in particular theinvention relates to DNA encoding the lethal genetic system of theExamples, in particular the modular transformation vector of Example 7herein, and to mutants and variants of such DNA having minor changessuch as substitutions, deletions or additions, but wherein the functionof the vector or lethal genetic system are not substantially affected,and the vector is able to cause the lethal effect of the invention asrequired.

[0122] Alternatively, multiple vectors may be used to transform theorganism with the necessary elements of the lethal system, if necessary.It is also possible that control elements and enhancers used to control,for example, a transcription factor which acts on the lethal gene, mayalso interfere with the lethal gene expression itself It may, therefore,be necessary to separate the components using silencer elements, orother genetic insulating elements to avoid unwanted gene expressionproblems.

[0123] The effect of a promoter or enhancer upon a gene normallyrequires the elements to be present on the same stretch of DNA However,the effect of a transcription factor may be exerted in trans, and may belocated on, for example, a different chromosome. The invention is notlimited to integration of the controlling elements on the samechromosome.

[0124] The construction of a recombinant multicellular organism mayrequire use of a transformation system for the target species (thespecies which is to be controlled). The specific nature of thetransformation system is not a critical feature of the invention, andtransformation protocols for a number of, for example, insects arealready known.

[0125] Vectors may be constructed using standard molecular biologytechniques in bacteria such as E. coli. We prefer that the vector usedfor transformation contains a selectable marker, such as genes producingG418 resistance or hygromycin resistance. Alternative genes other thanthose related to antibiotic resistance characteristics, such as greenfluorescent protein (GFP) may also be used. Expressed under the controlof a suitable promoter, this protein can be visualised simply byilluminating with a suitable excitatory wavelength (e.g. blue) andobserving the fluorescence. Such a marker would also allow easyidentification of trapped insects in release-and-recapture experiments.

[0126] Other suitable markers for transformation arc well known to theperson skilled in the art.

[0127] The invention also extends to cells, such as bacterial cells,transformed with a vector of the invention. Suitable cell lines formaintenance and/or propagation of such vectors, for example, are wellknown to the person skilled in the art.

[0128] We prefer that deletion of all or part of the lethal geneticsystem of the present invention from an organism gives no selectiveadvantage over an organism containing the system in permissiveconditions. The use of a lethal genetic system as described herein hassignificant advantages with respect to strain stability. In general,cross-mobilisation between related transposons and/or other unknownmechanisms can mean that trasposon insertions may not be as stable as“real” genes. When reared at a level of billions/week, as may berequired for biological control, even extremely rare events will happenrepeatedly. This is a major issue with the current medfly sexing strainswhere the chromosome translocations on which they depend break down (ata low frequency). Unfortunately, the resulting flies have significantlyhigher fitness than the rest of the stock and so their numbers tend toincrease rapidly. However, in the present system the breakdown product(deletion of all or part of the transposon) has no great advantage overthe intended stock, when reared on media containing Tc. Moreover, wherethere are multiple insertions, it would take several independent events(i.e. loss of each insertion), to make the stock completely ineffective.

[0129] If necessary, the lethal genetic complex may be furtherstabilised. Suitable methods include deleting one end of the transposonafter integration or secondary mobilisation of the system out of thetransposon into another site, using a site-specific recombination systemsuch as ERT/F1lp or cre/lox. Both of these systems are known to work inDrosophila.

[0130] The present invention will now be illustrated with respect to thefollowing Examples, which are for illustrative purposes only and are notlimiting upon the present invention, wherein:

[0131]FIG. 1 illustrates a modular vector for organism transformation;

[0132]FIG. 2 illustrates a model of a meiotic drive system of thepresent invention;

[0133]FIG. 3 illustrates a model of population control using multipleunlinked loci;

[0134]FIG. 4 illustrates a model of a meiotic drive system according tothe present invention;

[0135]FIG. 5 illustrates a model of population control using multipleunlinked loci;

[0136]FIG. 6 illustrates a further model of a meiotic drive system ofthe present invention;

[0137]FIG. 7 illustrates model of population control using multipleunlinked loci; and

[0138]FIG. 8 illustrates models of population control using theparameters of FIGS. 2, 6 and 7, but wherein the first two releases aredoubled in size.

EXAMPLES

[0139] Biological Control In a Drosophila Model

[0140] Introduction

[0141] In one specific embodiment, a two-part system may be used toproduce a conditional lethal effect. This system is based upon therepressor (tetR) of the transposon-1-derived tetracycline (Tc)resistance operon of E. coli. The use of this repressor for repressiblegene expression in eukaryotes has been developed by Manfred Gossen andHermann Bujard (reviewed in Gossen, et al., TIBS 18 471-475 1993). Inthis system, the tetR gene product is fused to the acidic domain ofVP16, to create a highly efficient Tc-repressible transactivator (tTA).

[0142] The first part of the system is the tTA expressed under thecontrol of a suitable promoter, and the second part is a dominant lethalgene expressed under the control of the tTA. Overall this givesexpression of the dominant lethal in a Tc-repressible fashion. Whentetracycline is not available, the tTA activates the lethal gene. Whentetracycline is present, it binds to the tTA and prevents activation ofthe lethal gene by tTA This lethal system is under the control of apromoter of choice. One further level of control can be exerted by thechoice of which Tc-analogue to use for repression: different analogueswill have different half-lives in the insect leading to induction of thekiller gene more or less promptly after the repressor is withdrawn fromthe diet.

[0143] We prefer that a non-bactericidal analogue should be used, so asnot to encourage tetracycline resistance in environmentalmicro-organisms. Use of a non-bactericidal analogue is in any caseessential for species such as tsetse fly, which have symbiotic bacteriaessential for reproduction of the fly which are killed by antibiotics.

[0144] Even this one system may be varied to provide a flexible tool forpopulation control. Greater flexibility may be achieved by combining twoor more promoters or enhancers. For example, medfly control might useexpression in the adult female (to prevent release of egg-layingfemales), and in early embryonic development (to prevent larval growthwithin the fruit). Since this means expression before the embryo startsto feed for itself, it would be important for growing the stock that arelatively stable Tc analogue is used, so that the embryos survivebecause of the maternal contribution of Tc. Larval expression could bealso used as an alternative, but with greater damage to the fruit.

[0145] Use of the above system to control the lethal effect of thelethal gene is only one example of how an effect could be achieved, andthere are numerous promoters, transactivator and lethal genes, forexample, which could be used to achieve the desired effect.

[0146] In the above scenario expression at more than one stage may berequired This could be achieved by using two separate tTA constructs, orby combining stage-specific enhancers into a single constructAppropriate promoters for stage-specific expression may be identified bysubtractive hybridisation or other known methods.

[0147] Insertion of the lethal gene or system into the chromosome of thetransgenic orgasm may be at any suitable point. It is not necessary todetermine the location of the lethal gene on the chromosome. Even thoughinserted elements may respond to control elements in adjacent chromatinthis not an issue for the tRE-killer lines, where lines givinginappropriate expression will probably not survive.

[0148] The present invention has been exemplified in the model insectspecies Drosophila melanogaster. Though D. melanogaster is not aneconomically important pest, it is experimentally tractable. The tTAsystem in general has been demonstrated in Drosophila (Bello, B., etal., 1998, Development 125:2193-2202). The Hsp26-tTA and tRE-lacZ usedbelow, and some vectors [described below], came from this paper.

[0149] Components:

[0150] Transactivator Component (Promoter-tTA)

[0151] Hsp26tTA: Heat shock protein 26-tTA Low basal level, heat-shockinducible to higher level, not sex-specific.

[0152] Obtained from Bruno Bello (NIMR, London). As detailed in Bello etal. (1998) Development 125, 2193-2202. Hsp26 promoter region with aportion of the translated region (sequences from -1917 to +490) wasfused to a tTa coding region isolated as an EcoRI/BamHI fragment frompUHD 15-1. neo followed by the transcription termination sequence of theHsp70 gene.

[0153] Act5C-tTA: Actin 5C-tTA. Strong, constitutive, ubiquitouspromoter, not sex-specific.

[0154] The tTA coding region was excised as an EcoRT/PvuII fragment thenend filled using T4 DNA polymerase. The p CaSpeR {Actin5C GFP}(Reichhart and Ferrandon, (1998), D. S. 81: 201-202) was digested withXbaI/Ba to remove the GFP fragment than end filled using T4 polymerase.These two fragments were then ligated. The resulting clones werescreened using a SmaI/EcoRV digest to select a clone of the correctorientation, placing the tTa coding region under the control of theActin 5C promoter.

[0155] Stwl-tTA: Stonewall-tTa Female-specific in embryos, but expressedlater in both sexes.

[0156] The tTa coding region was excised from the plasmid pUHD 15-1.noby digestion with EcoRI and PvuII. This fragment was then ligated intothe vector pstwl^(+mCa) (Clark, K A. and McKearin D. M. (1996),Development 122 (3): 937-950) digested with EcoRI/PvuII such that tTawas placed under the transcriptional control of 1.7 kb of stwl promotergenomic DNk

[0157] Sxl^(pe)-tTA: Sex lethal-tTA. Early promoter (PE) from Sxl.Thought to be expressed in early female embryos only.

[0158] The tTa coding region was excised from the plasmid pUHD 15-1.neo(Gossen M. and Bujard H. (1992); PNAS, 89, 5547-51) by digestion withEcoRI/PuvII. This fragment was then ligated into the 5-1 sxl^(pe):bluescript (containing Sxl^(pe) sequences (Keyes L N, et al. (1992)Cell. 6; 68(5): 933-43) digested with EcoRI and EcoRV to create sxlpetTa bluescript. A KpnI/NotI fragment containing the tTa coding regionand sxlpe promoter was subcloned into the P element transformationvector pP {W8} (Klemenz et al., (1987) Nucleic Acids Res. 15: 3947-3959)digested with KpnI/NotI to create p(sxl^(pe)tTa).

[0159] Yp3-tTA Yolk protein 3-tTA. Female fat body enhancer ABE) fromyolk protein 3, with hsp70 minimal promoter. Expressed in female fatbody in larvae and adults.

[0160] The tTa coding region was excised from the plasmid pUHD 15-1.neoby digestion with EcoRI and PvuII. This fragment was then cloned betweenthe EcoRT/PvuII sites of the yp 3 expression construct pFBE (Bownes M,personal communication) such that it was under the transcriptionalcontrol of the Female Fat Body Enhancer (FBE) Ronaldson E, et al., GenetRes. 1995 Aug; 66(1): 9-17.) and a minimal viral promoter.

[0161] tRE-responsive gene

[0162] tRe-lacZ: E. coli lacZ gene, encoding β-galactosidase. Used asreporter. Obtained from Bruno Bello (N London) As detailed in Bello etal.(1998) Development 125, 2193-2202. The heptameric repeat of the tetoperator was isolated as a EcoRI/KpnI fragment from pUHC 13-3 (Gossen M.and Bujard H. (1992); PNAS, 89,5547-51) and cloned upstream of theP-lacZ fusion of the enhancer-test vector CPLZ (Wharton K A and Crews ST. (1994) Development. 120(12): 3563-9.). CPLZ contain5 the P elementtransposase promoter (up to —42 from cap site) and the N-terminaltransposase sequence fused in-frame with lacz and the polyadenylationsignal of SV40.

[0163] WTP-2 (white-tetO-P promoter—vector containing tRe sequences)

[0164] Obtained from Bruno Bello (NIMR, London). As detailed in Bello etal. (1998) Development 125, 2193-2202. This P-element vector wasconstructed to express any gene under the control of atetracycline-responsive promoter. It contains the vector backbone ofCPLZ, the heptameric repeat of the tet operator, the P-element promoterand leader sequences from Carnegie 4 (Rubin G M and Spradling A C (1983)Nucleic Acids Res Sep 24; 11(18): 6341-51) and the polyadenylationsignal of SV40.

[0165] WTP-3 (modified WTP-2)

[0166] The WTP-2 vector was modified by the addition of two complimentshort oligos 5′ UAS ATG+(AATTGCCACCATGGCTCATATGGAATTCAGATCTG) and 3′ UASATG (GGCCGCAGATCTGAATTCCATATGAGCCATGGTGGGC) into the WTP-2 MCS. Theoligos were allowed to anneal and ligated to WTP-2 digested withEcoRI/NotI. These oligos introduced a consensus translation standseveral additional cloning sites into the WTP-2 multiple cloning site(MCS).

[0167] tRe-EGFP. Encodes a mutant version of Green Fluorescent Protein(GFP), a jellyfish (Aequoria) gene encoding a fluorescent protein. TheEGFP mutant has two amino acid changes, giving a brighter, more solubleprotein Used as a reporter. The enhanced green fluorescent protein(EGFP, a F64L, S65T mutant derivative of GFP) coding region (Craven etal. (1998) Gene 9; 221(1): 59-68) was isolated as a NcoI/EcoRI fragmentfrom the pP{UAS-EGFP) vector, then end filled with T4 polymerase. pP{UAS-EGFP}. pP {UAS-EGFP} was constructed as follows.

[0168] The single NdeI site of pP{UAST} was eliminated by digestion,end-filling and re-ligation, in order to be able to use NdeI in themultiple cloning sites. We then used two oligonucleotides (USA-ATG+=5′AATTGCCACCATGGCTCATATGGAATTCAGATCTGC and UAS-ATG-=5′GGCCGCAGATCTGAATTCCATATGAGCCATGGTGGC), allowed them to anneal ligatedthem to EcoRI-NotI digested pP {UAST} {from which the Ndel site had beenremoved} to make pP {UAS-LP}. We amplified inserts from pGEM-T-EGFP[Craven, 1998, supra] using Pfu polymerase and the oligonucleotides 5′TAGGAGTAAAGGAGAAGAAC and 5′ AATTCCATATGTTTGTATAGTTCA. Each PCR productwas gel-purified then incubated with T4 DNA polymerase in the presenceof dGTP and dCTP but not dATP or dTTP. This created an NdeI-compatiblecohesive end at one end of the fragment and an EcoRI-compatible cohesiveend at the other end. These fragments were then subcloned intoNdeI-EcoRI digested pP {UAS-LP}pP { UAS-EGFP}.

[0169] The WTP-3 vector was then digested with EcoRI and end filled withT4 polymerase and the fragments ligated together. A diagnostic digestusing PvuII/BamHI was then used to select a clone of the correctorientation.

[0170] tRe-Ras64BV32. Mutant version of Drosophila melanogaster Ras64B,involved in cell signalling. Mutant is constitutively active, making ittoxic to the cell if expressed at a high enough level. Toxicity is notsex-specific. The Ras₆₄B^(V12) cDNA was cloned as an EcoRI/NotI fragmentfrom the p {sevRas64B^(V12)}(Matsuo et al., (1997), Development 124(14):2671-2680), into WTP-2 digested with EcoRI/NotI.

[0171] tRe-Mls-^(Mpu). Mutant version of Drosophila melanogaster Msl-1.Msl-1 is a component of the sex determination pathway that is usuallyexpressed only in males, being repressed in females by a product of theSex lethal gene. Activity of mutant is independent of Sex lethal, makingit toxic to females if expressed at a high enough level. Toxicity istherefore sex-specific. The msl-1^(MPU) cDNA was cloned as an EcoRIfragment from M1-ECTOPIC (Chang and Kuroda, (1998) Genetics 150(2):699-709) into the WTP-2 vector digested with EcoRI A diagnostic digestusing HindIII/NotI, was then used to select a clone of the correctorientation, placing the msl-1^(MPU) cDNA under the control of the tResequences.

[0172] tRe-Ms-2Nopu. Mutant version of Drosophila melanogaster Msl-2.Msl-² is another component of the sex determination pathway that isusually expressed only in males, being repressed in females by a productof the Sex lethal gene. Activity of mutant is independent of Sex lethal,making it toxic to females if expressed at a high enough level. Toxicityis therefore sex-specific. The msl-2 cDNA was cloned as a NotI/XbaIfragment from pM2 NOPU (Kelley et al., (1995), Cell 81; 867-877) andcloned into WTP-2 digested with NotI/XbaI.

Example 1

[0173] Single Chromosome Crossos

[0174] In “single chromosome crosses” at 25° C., ten to fifteen virginfemales homozygous for the tTA construct and five to ten young maleshomozygous for the tRe construct were placed on food containing orlacking a tetracycline supplement. Their progeny were allowed to developon this food. Tetracycline conc. μg/ml Female Total Male Total 0 0^(A),0^(B), 0^(C), 0^(F), 0, 0, 0, 0 0 58, 47, 60, 51, 46, 60, 52, 54 428 0.146, 49, 50, 51, 52, 50, 41, 40 379 56, 42, 72, 41, 56, 72, 61, 34 434 152, 40, 60, 0, 60, 72, 50, 52 386 50, 51, 55, 3, 63, 54, 57, 56 389 541, 55, 49, 52, 48, 47, 40, 51 383 36, 47, 42, 55, 36, 55, 52, 52 375

[0175] Sxl^(pe)

[0176] Format for data: the 8 numbers are the results from crosses usingindependent insertions of each element (to control for position effect).Here, 4 insertions of Sxl^(pe)-tTA (A, B, C, and F) were used and two oftRE-Ras 64B^(V12) (B and C). The order of the data are:Sxl^(pe)-tTA^((A)) females with tRe Ras64B^(V12(B)) males, tenSxlB×RasB, SxlC×RasB, SxIF×RasB, SxlA×RasC, SxIB×RasC, SxlC×RasC andfinally SxlF×RasC. Data are presented in a similar fashion in the othertables Tetracycline conc. μg/ml Female Total Male Total 0 0, 0, 0, 0, 0,0, 0, 0 0 59, 57, 62, 51, 73, 69, 57, 483 55 0.1 61, 52, 47, 46, 22, 31,36, 15 296 60, 62, 56, 71, 69, 75, 55, 520 72 1 59, 57, 63, 59, 31, 21,15, 21 326 47, 56, 49, 62, 63, 67, 71, 473 58 5 61, 47, 52, 56, 38, 22,16, 12 304 68, 72, 67, 92, 58, 54, 61, 535 63

[0177] Tetracycline conc. μg/ml Female Total Male Total 0 0, 0, 0, 0, 0,0, 0, 0, 0, 0, 0, 0 0 56, 72, 81, 69, 62, 63, 56, 761 47, 82, 57, 55, 610.1 79, 56, 47, 42, 51, 61, 52, 52, 647 58, 41, 40, 35, 50, 67, 71, 56249, 51, 53, 54 39, 52, 62, 40, 70 1 42, 45, 56, 48, 52, 61, 57, 54, 64460, 39, 61, 60, 69, 49, 59, 674 55, 56, 57, 61 38, 64, 69, 71, 35 5 58,61, 52, 53, 54, 61, 29, 31, 615 61, 59, 57, 56, 55, 48, 91, 742 55, 50,49, 62 63, 54, 50, 81, 67

[0178] Tetracycline conc. μg/ml Female Total Male Total 0 0, 0, 0, 0, 0,0 0 0, 0, 0, 0, 0, 0 0 0.1 36, 62, 71, 41, 49, 58 317 43, 442, 63, 35,68 315 1 58, 37, 58, 41, 55, 58 307 47, 70, 51, 51, 39, 70 328 5 36, 38,56, 43, 34, 64 271 57, 71, 68, 53, 44, 42 335

[0179] Tetracycline conc. μg/ml Female Total Male Total 0 0, 0, 0, 0, 0,0 0 50, 44, 45, 56, 40, 67 302 0.1 67, 56, 37, 23, 16, 12 211 56, 53,50, 61, 42, 74 336 1 69, 64, 41, 13, 31, 18 236 33, 70, 39, 45, 40, 70257 5 52, 42, 49, 19, 20, 41 223 37, 80, 41, 48, 80 291

[0180] Tetracycline conc. μg/ml Female Total Male Total 0 0, 0, 0, 0, 0,0, 0, 0, 0 0 38, 53, 47, 68, 38, 70, 52, 60, 481 55 0.1 54, 57, 41, 64,40, 63, 39, 436 59, 58, 49, 73, 48, 69, 45, 47, 491 42, 36 43 1 46, 34,35, 63, 47, 70, 64, 439 55, 40, 40, 71, 50, 72, 74, 46, 490 39, 41 42 552, 70, 37, 34, 35, 57, 49, 434 54, 71, 41, 42, 41, 66, 56, 55, 481 50,50 55

[0181] Tetracycline conc. μg/ml Female Total Male Total 0 0, 0, 0, 0, 0,0 0 0, 0, 0, 0, 0, 0 377 0.1 77, 57, 69, 50, 45, 63 361 50, 70, 71, 67,53, 61 372 1 86, 59, 60, 80, 70, 72 427 46, 89, 72, 45, 76, 55 383 5 46,49, 87, 63, 59, 71 375 75, 83, 58, 83, 72, 82 400

[0182] Tetracycline conc. μg/ml Female Total Male Total 0 0, 0, 0, 0, 0,0 0 83, 73, 65, 69, 53, 80 423 0.1 72, 74, 80, 68, 72, 46 412 82, 52,57, 66, 86, 59 402 1 61, 83, 48, 66, 65, 57 321 74, 69, 85, 58, 48, 61351 5 70, 57, 50, 62, 61, 86 386 48, 68, 52, 62, 84, 87 401

[0183] Tetracycline conc. μg/ml Female Total Male Total 0 0, 0, 0, 0, 0,0, 0, 0, 0 0 63, 52, 67, 71, 88, 55, 46, 86, 75 603 0.1 84, 85, 83, 73,48, 48, 46, 71, 58 548 62, 54, 48, 81, 85, 74, 78, 77, 78 637 1 70, 70,66, 81, 50, 52, 69, 81, 51 590 69, 87, 47, 64, 66, 59, 58, 47, 52 549 567, 70, 87, 61, 54, 54, 67, 74, 81 615 71, 61, 57, 53, 51, 65, 45, 68,51 522

[0184] Tetracycline conc. μg/ml Female Total Male Total 0 0, 0, 0, 0 00, 0, 0, 0 0 0.1 47, 56, 71, 61 235 46, 52, 53, 59 210 1 60, 46, 52, 41199 79, 71, 68, 56 274 5 2, 51, 71, 32 156 0, 49, 62, 43 154

[0185] Tetracycline conc. μg/ml Female Total Male Total 0 0, 0, 0, 0, 0,0 0 64, 58, 33, 66, 55, 42 318 0.1 45, 44, 72, 56, 62, 49 328 53, 54,80, 57, 66, 58 368 1 70, 35, 61, 50, 57, 37 310 78, 36, 70, 56, 61, 42343 5 44, 58, 58, 59, 42, 52 313 46, 68, 66, 64, 48, 55 347

[0186] Tetracycline conc. μg/ml Female Total Male Total 0 0, 0, 0 0 56,47, 56 159 0.1 48, 49, 62 159 56, 68, 49 159 1 43, 45, 51 135 36, 39, 47122 5 55, 3, 66 124 61, 5, 54 120

[0187] Tetracycline conc. μg/ml Female Total Male Total 0 0, 0, 0, 0, 0,0 0 65, 70, 61, 65, 47, 42 350 0.1 33, 54, 50, 72, 63, 50 322 42, 64,52, 74, 67, 54 352 1 56, 56, 61, 69, 57, 43 342 59, 64, 65, 75, 64, 49376 5 46, 51, 73, 65, 42, 39 316 44, 56, 79, 74, 52, 49 354

[0188] Tetracycline conc. μg/ml Female Total Male Total 0 0, 0, 2, 0, 0,0 2 49, 58, 39, 65, 35, 51 297 0.1 36, 65, 71, 37, 59, 68 336 46, 73,77, 46, 66, 71 379 1 42, 65, 67, 57, 35, 53 319 49, 72, 68, 59, 41, 58347 5 55, 55, 43, 58, 36, 60 307 63, 64, 49, 63, 45, 64 348

[0189] Tetracycline conc. μg/ml Female Total Male Total 0 0, 0, 0, 0, 0,0 0 35, 35, 72, 52, 45, 37 276 0.1 34, 68, 42, 51, 33, 40 268 35, 72,45, 56, 36, 44 248 1 41, 39, 42, 60, 70, 72 324 51, 49, 46, 61, 78, 77362 5 70, 55, 56, 65, 43, 61 349 74, 58, 64, 73, 51, 66 386

[0190] Conclusion

[0191] These data show that one or both sexes can be efficientlyeliminated, while good repression of this lethality can be achieved bythe addition of modest concentrations of tetracycline to the food. Thisrepression is effective over a wide range of tetracyclineconcentrations.

Example 2

[0192] Reporter Crosses

[0193] In “reporter crosses” at 25° C., females homozygous carrying aninsertion of Sxlp^(e) tTa on their X chromosome (Sxlp^(e) tTa^((A)))were crossed to males carrying various reporter constructs. As with“single chromosome crosses”, ten to fifteen virgin females homozygousfor the tTA construct and five to ten young males homozygous for the tReconstruct were placed on food containing or lacking a tetracyclinesupplement. Their progeny were allowed to develop on this food.

[0194] lacZ

[0195] Embryos were stained for IacZ using a standard histochemicalmethod. Tetracycline conc. μg/ml LacZ positive Total LacZ negative Total0 60, 85, 99, 60 304 78, 89, 85, 93 345 0.1 0, 0, 0, 0 0 176, 174, 178,181 709 1 0, 0, 0, 0 0 188, 190, 181, 180 739 5 0, 0, 0, 0 0 156, 151,159, 185 651

[0196] Tetracycline conc. μg/ml LacZ positive Total LacZ negative Total0 57, 82, 97, 45 281 61, 74, 59, 82 276 0.1 0, 0, 0, 0 0 131, 165, 132,90 518 1 0, 0, 0, 0 0 170, 161, 181, 195 707 5 0, 0, 0, 0 0 126, 190,190, 196 702

[0197] Tetracycline conc. μg/ml LacZ positive Total LacZ negative Total0 0, 0, 0, 0 0 189, 200, 153, 169 711 0.1 0, 0, 0, 0 0 164, 175, 190,179 708 1 0, 0, 0, 0 0 182, 190, 195, 167 737 5 0, 0, 0, 0 0 199, 151,169, 164 683

[0198] EGFP

[0199] Embryos were scored for fluorescence. In the case of embryos ontetracycline-free media, these were separated, allowed to develop ontetracycline-free media and the sex of the emerging adults was scored.Tetracycline conc. μg/ml Fluorescent female male Non-Fluorescent femalemale 0 89, 100, 53, 55 200 0 99, 86, 46, 51 0 232 0.1 0, 0, 0, 0 — —199, 182, 188, 153 — — 1 0, 0, 0, 0 — — 170, 135, 163, 196 — — 5 0, 0,0, 0 — — 186, 159, 127, 200 — —

[0200] Tetracycline conc. μg/ml Fluorescent female male Non-Fluorescentfemale male 0 60, 91, 62, 83 243 0 102, 56, 79, 72 1 256 0.1 0, 0, 0, 0— — 196, 170, 165, 162 — — 1 0, 0, 0, 0 — — 182, 200, 197, 161 — — 5 0,0, 0, 0 — — 182, 161, 188, 182 — —

[0201] Tetracycline conc. μg/ml Fluorescent male female Non-Fluorescentmale female 0 0, 0, 0, 0 — — 196, 179, 165, 164 — — 0.1 0, 0, 0, 0 — —179, 197, 198, 188 — — 1 0, 0, 0, 0 — — 198, 187, 190, 164 — — 5 0, 0,0, 0 — — 170, 177, 199, 165 — —

[0202] C(1) DX is a compound X chromosome; effectively two X chromosomesjoined together. The X chromosome from males crossed to C(1)DX femalesis therefore inherited by the sons, rather than the daughters.

[0203] Conclusions

[0204] The data demonstrate that, as expected, reporter gene expressionis turned off in the presence of over a range of concentrations.

Example 3

[0205] Recombinant Chromesome Experiments

[0206] 40-50 young females and 20-25 young males raised at 25° C. uponfood with the indicated tetracycline supplement were allowed to mate,then transferred to normal (tetracycline-free) food after 3-4 days.These flies were transferred to fresh vials of normal food every day for12 days, then moved on the 13th day. All the vials were incubated at 25°C. while the progeny developed. The numbers of male and female progenyemerging as adults in each vial were recorded.

Tetracycline Concentration

[0207] Sxl^(pe) Tet. Conc. Day 1 Fe- Day 2 Day 3 Fe- Day 4 Day 5 Day 6Day 7 μg/ml Male male Male Female Male male Male Female Male Female MaleFemale Male Female 0.1 103 0 98 0 89 0 92 0 105 0 95 0 110 0 1 128 0 1370 150 0 136 0 111 0 87 0 100 0 5 110 0 111 0 95 0 90 0 144 0 93 0 138 020 131 0 126 0 133 0 120 0 93 0 99 0 111 0 100 139 0 127 0 145 0 110 0149 0 128 0 94 0 500 95 11 133 12 145 1 137 1 86 0 112 0 128 0 1000 14012 133 24 119 8 94 2 92 1 137 1 129 1 2000 110 35 97 25 94 16 138 12 1152 126 1 145 1 Day 8 Day 9 Day 10 Day 11 Day 12 Total Tet. Conc. μg/mlMale Female Male Female Male Female Male Female Male Female Male Female0.1 106 0 131 0 148 0 86 0 99 0 1262 0 1 106 0 109 0 97 0 124 0 114 01399 0 5 106 0 89 0 148 0 148 0 87 0 1359 0 20 87 0 149 0 104 0 113 0132 0 1398 0 100 93 0 125 0 99 0 121 0 139 0 1459 0 500 142 0 129 0 1140 131 0 126 0 1478 25 1000 89 0 94 0 97 0 138 0 87 0 1349 49 2000 94 0137 0 99 0 141 0 143 0 1439 92

[0208] Tet. Conc. Day 1 Fe- Day 2 Day 3 Fe- Day 4 Day 5 Day 6 Day 7μg/ml Male male Male Female Male male Male Female Male Female MaleFemale Male Female 0.1 103 0 98 0 149 0 121 0 134 0 150 0 117 0 1 149 086 0 111 0 112 0 126 0 148 0 136 0 5 104 0 99 0 148 0 128 0 142 0 134 093 0 20 121 0 106 0 97 0 127 0 142 0 131 0 107 0 100 94 0 142 0 115 0131 0 114 0 103 0 131 0 500 140 34 148 23 100 14 95 1 122 0 120 0 115 01000 110 29 87 12 138 22 145 17 91 5 106 1 102 1 2000 123 42 145 37 13143 139 15 126 12 118 7 100 4 Day 8 Day 9 Day 10 Day 11 Day 12 Total Tet.Conc. μg/ml Male Female Male Female Male Female Male Female Male FemaleMale Female 0.1 138 0 142 0 147 0 130 0 112 0 1541 0 1 129 0 123 0 91 099 0 131 0 1441 0 5 99 0 106 0 95 0 144 0 129 0 1421 0 20 149 0 150 0 890 128 0 140 0 1487 0 100 93 0 119 0 143 0 87 0 144 0 1416 0 500 98 0 1290 90 0 124 0 107 0 1388 72 1000 92 0 150 0 145 0 107 0 143 0 1416 872000 92 1 120 0 89 0 106 0 149 0 1438 161

[0209] Tet. Conc. Day 1 Fe- Day 2 Day 3 Fe- Day 4 Day 5 Day 6 Day 7μg/ml Male male Male Female Male male Male Female Male Female MaleFemale Male Female 0.1 97 0 136 0 152 0 130 0 108 0 114 0 88 0 1 102 099 0 134 0 171 0 171 0 118 0 91 0 5 130 0 159 0 158 0 91 0 84 0 127 0110 0 20 76 0 129 0 128 0 79 0 89 0 98 0 94 0 100 112 0 145 0 130 0 1240 79 0 109 0 134 0 500 136 2 79 0 161 0 102 0 171 0 151 0 161 0 1000 9215 83 9 150 3 149 2 146 0 92 0 115 0 2000 127 21 95 14 153 3 164 4 135 197 1 144 0 Day 8 Day 9 Day 10 Day 11 Day 12 Total Tet. Conc. μg/ml MaleFemale Male Female Male Female Male Female Male Female Male Female 0.1140 0 104 0 141 0 173 0 81 0 1464 0 1 104 0 120 0 171 0 102 0 144 0 15270 5 116 0 123 0 155 0 163 0 121 0 1535 0 20 122 0 103 0 126 0 123 0 78 01243 0 100 127 0 133 0 79 0 157 0 154 0 1483 0 500 164 0 95 0 160 0 1540 91 0 1625 2 1000 168 0 153 0 80 0 95 0 79 0 1402 29 2000 158 0 103 0129 0 141 0 97 0 1543 44

[0210] Tet. Conc. Day 1 Fe- Day 2 Day 3 Fe- Day 4 Day 5 Day 6 Day 7μg/ml Male male Male Female Male male Male Female Male Female MaleFemale Male Female 0.1 111 0 108 0 130 0 69 0 101 0 110 0 130 0 1 89 0106 0 119 0 70 0 87 0 117 0 138 0 5 112 0 80 0 68 0 130 0 78 0 93 0 78 020 92 0 83 0 129 0 127 0 66 0 69 0 95 0 100 72 0 90 0 72 0 66 0 106 0122 0 100 0 500 78 0 118 0 69 0 67 0 88 0 83 0 135 0 1000 122 2 107 1133 0 116 0 115 0 107 0 119 0 2000 134 12 79 14 123 5 130 1 102 0 114 083 0 Day 8 Day 9 Day 10 Day 11 Day 12 Total Tet. Conc. μg/ml Male FemaleMale Female Male Female Male Female Male Female Male Female 0.1 127 0 920 79 0 77 0 133 0 1267 0 1 71 0 104 0 81 0 124 0 85 0 1171 0 5 106 0 840 135 0 119 0 82 0 1165 0 20 101 0 71 0 108 0 74 0 112 0 1127 0 100 1360 104 0 116 0 77 0 107 0 1168 0 500 128 0 104 0 73 0 106 0 88 0 1137 01000 101 0 115 0 86 0 96 0 92 0 1309 3 2000 130 0 105 0 120 0 104 0 1010 1325 32

[0211] Tet. Conc. Day 1 Fe- Day 2 Day 3 Fe- Day 4 Day 5 Day 6 Day 7μg/ml Male male Male Female Male male Male Female Male Female MaleFemale Male Female 0.1 93 0 137 0 84 0 66 0 114 0 107 0 114 0 1 73 0 900 99 0 120 0 118 0 85 0 85 0 5 84 0 122 0 131 0 93 0 104 0 90 0 133 0 20127 0 128 0 80 0 105 0 81 0 122 0 108 0 100 72 0 80 0 87 0 128 0 78 0 920 88 0 500 98 0 78 0 94 1 105 0 138 0 77 0 92 0 1000 117 1 105 2 130 1130 1 82 0 88 0 113 0 2000 91 16 70 11 69 13 70 4 108 1 90 0 115 0 Day 8Day 9 Day 10 Day 11 Day 12 Total Tet. Conc. μg/ml Male Female MaleFemale Male Female Male Female Male Female Male Female 0.1 117 0 78 0123 0 125 0 121 0 1279 0 1 91 0 90 0 68 0 88 0 82 0 1089 0 5 89 0 70 0138 0 85 0 100 0 1239 0 20 95 0 118 0 70 0 114 0 114 0 1262 0 100 66 0137 0 85 0 109 0 93 0 1113 0 500 68 0 70 0 109 0 86 0 136 0 1151 1 100095 0 137 0 99 0 120 0 66 0 1282 5 2000 84 0 98 0 83 0 128 0 131 0 113745

[0212] Hsp26 Tet. Conc. Day 1 Fe- Day 2 Day 3 Fe- Day 4 Day 5 Day 6 Day7 μg/ml Male male Male Female Male male Male Female Male Female MaleFemale Male Female 0.1 153 0 154 0 127 0 130 0 81 0 151 0 147 0 1 138 098 0 74 0 68 0 150 0 123 0 115 0 5 140 0 132 0 119 0 129 0 87 0 156 0157 0 20 115 0 113 0 92 0 92 0 129 0 77 0 119 0 100 150 0 127 0 126 0114 0 78 0 93 0 98 0 500 119 1 146 0 154 0 132 0 112 0 97 0 80 0 1000 775 109 2 105 2 85 0 84 0 127 0 91 0 2000 156 18 101 6 149 3 115 1 134 0139 0 151 0 Day 8 Day 9 Day 10 Day 11 Day 12 Total Tet. Conc. μg/ml MaleFemale Male Female Male Female Male Female Male Female Male Female 0.1117 0 81 0 106 0 135 0 141 0 1523 0 1 152 0 89 0 105 0 146 0 89 0 1367 05 79 0 148 0 120 0 92 0 119 0 1478 0 20 69 0 78 0 149 0 72 0 116 0 12210 100 121 0 126 0 157 0 141 0 143 0 1474 0 500 142 0 103 0 104 0 144 0129 0 1462 1 1000 75 0 147 0 105 0 97 0 123 0 1225 9 2000 86 0 97 0 98 0131 0 76 0 1433 28

[0213] Tet. Conc. Day 1 Fe- Day 2 Day 3 Fe- Day 4 Day 5 Day 6 Day 7μg/ml Male male Male Female Male male Male Female Male Female MaleFemale Male Female 0.1 120 0 87 0 127 0 115 0 121 0 80 0 100 0 1 84 0153 0 100 0 88 0 93 0 71 0 126 0 5 134 0 95 0 122 0 141 0 80 0 77 0 1060 20 135 0 137 0 140 0 135 0 107 0 141 0 89 0 100 146 1 146 0 82 0 106 0118 0 118 0 82 0 500 124 12 144 8 99 2 154 1 137 0 96 1 75 0 1000 72 2785 17 76 15 87 12 102 5 93 5 69 3 2000 132 67 96 45 119 38 135 35 104 2290 17 149 12 Day 8 Day 9 Day 10 Day 11 Day 12 Total Tet. Conc. μg/mlMale Female Male Female Male Female Male Female Male Female Male Female0.1 151 0 79 0 108 0 69 0 107 0 1264 0 1 78 0 95 0 105 0 112 0 154 01259 0 5 135 0 84 0 152 0 145 0 142 0 1413 0 20 79 0 157 0 92 0 73 0 1390 1424 0 100 96 0 135 0 86 0 106 0 157 0 1378 1 500 139 0 142 0 145 0 840 136 0 1475 24 1000 114 1 145 0 130 0 136 0 152 0 1261 85 2000 149 2 810 127 0 146 0 88 0 1416 238

[0214] Yp3 Tet. Conc. Day 1 Fe- Day 2 Day 3 Fe- Day 4 Day 5 Day 6 Day 7μg/ml Male male Male Female Male male Male Female Male Female MaleFemale Male Female 0.1 93 0 119 0 112 0 141 0 100 0 126 0 89 0 1 117 0135 0 122 0 121 0 127 0 101 0 136 0 5 112 0 116 0 128 0 111 0 136 0 1130 130 0 20 89 0 107 0 107 0 98 0 88 0 102 0 107 0 100 129 0 136 0 128 0127 0 135 0 144 0 107 0 500 136 2 88 0 113 0 113 0 87 0 94 0 109 0 1000107 13 140 5 110 0 141 0 98 0 129 0 88 0 2000 119 32 102 15 107 12 109 9109 8 140 2 127 0 Day 8 Day 9 Day 10 Day 11 Day 12 Total Tet. Conc.μg/ml Male Female Male Female Male Female Male Female Male Female MaleFemale 0.1 105 0 133 0 93 0 131 0 121 0 1353 0 1 90 0 119 0 84 0 98 0100 0 1360 0 5 119 0 96 0 88 0 144 0 91 0 1384 0 20 135 0 126 0 143 0123 0 141 0 1366 0 100 96 0 92 0 104 0 94 0 115 0 1407 0 500 141 0 144 0123 0 104 0 124 0 1376 2 1000 138 0 105 0 124 0 115 0 114 0 1409 18 2000114 0 123 0 132 0 115 0 107 0 1404 78

[0215] Tet. Conc. Day 1 Fe- Day 2 Day 3 Fe- Day 4 Day 5 Day 6 Day 7μg/ml Male male Male Female Male male Male Female Male Female MaleFemale Male Female 0.1 121 0 94 0 103 0 93 0 96 0 119 0 119 0 1 95 0 1230 79 0 78 0 130 0 103 0 112 0 5 109 0 110 0 118 0 124 0 86 0 122 0 90 020 81 0 89 0 127 0 82 0 81 0 79 0 128 0 100 112 0 87 1 87 1 113 0 95 191 1 84 1 500 84 21 96 15 86 15 124 9 123 5 86 3 106 1 1000 100 47 11012 109 6 103 13 102 9 97 2 82 6 2000 127 63 130 54 128 34 117 21 89 1287 11 90 4 Day 8 Day 9 Day 10 Day 11 Day 12 Total Tet. Conc. μg/ml MaleFemale Male Female Male Female Male Female Male Female Male Female 0.184 0 127 0 104 0 76 0 95 0 1231 0 1 94 0 106 0 83 0 93 0 113 0 1209 0 5132 0 126 0 76 0 128 0 102 0 1323 0 20 119 0 99 0 90 0 106 0 87 0 1168 0100 85 1 122 0 114 0 90 0 126 0 1206 6 500 85 1 93 0 111 0 111 0 104 01209 71 1000 95 0 113 0 110 0 85 0 87 0 1193 97 2000 131 1 128 0 91 0 950 82 0 1295 200

[0216] Tet. Conc. Day 1 Fe- Day 2 Day 3 Fe- Day 4 Day 5 Day 6 Day 7μg/ml Male male Male Female Male male Male Female Male Female MaleFemale Male Female 0.1 91 0 84 0 107 0 80 0 88 0 92 0 89 0 1 117 0 92 0128 0 80 0 104 0 116 2 8 0 5 82 0 123 0 116 0 120 2 89 0 90 0 95 0 20 921 101 0 87 0 109 0 81 0 121 0 83 1 100 108 13 130 9 131 5 99 7 109 3 1231 107 1 500 78 22 85 16 80 12 106 15 130 11 91 10 118 7 1000 130 35 8642 78 26 116 14 80 12 82 17 77 15 2000 116 79 130 72 78 44 101 29 132 3294 22 89 16 Day 8 Day 9 Day 10 Day 11 Day 12 Total Tet. Conc. μg/ml MaleFemale Male Female Male Female Male Female Male Female Male Female 0.188 1 135 0 123 0 120 0 114 0 1229 1 1 101 0 127 2 84 0 101 0 79 0 1137 45 80 0 94 0 127 0 128 3 86 0 1230 5 20 132 0 81 0 88 0 112 0 127 0 12142 100 106 1 132 0 81 0 115 0 107 0 1348 40 500 115 2 98 0 86 0 82 4 1150 1184 99 1000 131 3 104 1 99 0 125 0 108 0 1216 165 2000 91 8 88 2 85 5114 0 80 0 1198 309

[0217] Conclusion

[0218] These data show that feeding the mothers high concentrations oftetracycline has some protective effect, but that all these recombinantchromosomes work extremely efficiently over a wide range of (parental)tetracycline concentrations, with the sole exception of “Yp3 tTa, tReMsl-1^(Mpu) on the 2^(nd) chromosome”, which has some (<1%) escaperseven at low tetracycline concentrations. Since there is no meioticrecombination in Drosophila melanogaster males, any of these recombinantchromosomes could be used in a genetic sexing or insect control program,if required. In practice, Drosophila melanogaster is not an agriculturalpest or disease vector, but these data demonstrate that the effectiveelimination of one sex can be achieved by this method.

Example 4

[0219] Use of Non-antibiotic Tetracycline Analogues

[0220] Recombinant chromosome stocks can readily be maintained at 25° C.on epioxytetracyclie concentrations of 1 μg/ml or anhydrotetracyclineconcentrations of 0.1 μg/ml, showing that these non-antibiotictetracycline analogues are effective in repressing tTA responsive geneexpression.

[0221] Epioxytetracycline

[0222] A standard range of additive concentrations were used in thefollowing experiments (0.05-20 μg/ml). We were unable to maintain stockat two lowest concentrations, so marked n.d. (=“not done ”)Epioxytetracycline Conc. μg/ml Female Male 0.05 n.d. n.d. 0.1 n.d. n.d.1 0 1306 5 0 1581 20 0 1495

[0223] Epioxytetracycline Conc. μg/ml Female Male 0.05 n.d. n.d. 0.1n.d. n.d. 1 0 1165 5 0 1279 20 0 1257

[0224] Epioxytetracycline Conc. μg/ml Female Male 0.05 n.d. n.d. 0.1n.d. n.d. 1 0 1076 5 0 1119 20 0 1159

[0225] Epioxytetracycline Conc. μg/ml Female Male 0.05 n.d. n.d. 0.1n.d. n.d. 1 0 1250 5 0 1300 20 0 1364

[0226] Epioxytetracycline Conc. μg/ml Female Male 0.05 n.d. n.d. 0.1n.d. n.d. 1 0 1483 5 0 1585 20 0 1565

[0227] Epioxytetracycline Conc. μg/ml Female Male 0.05 n.d. n.d. 0.1n.d. n.d. 1 0 1362 5 0 1181 20 0 1403

[0228] Epioxytetracycline Conc. μg/ml Female Male 0.05 n.d. n.d. 0.1n.d. n.d. 1 0 1243 5 0 1409 20 0 1373

[0229] Epioxytetracycline Conc. μg/ml Female Male 0.05 n.d. n.d. 0.1n.d. n.d. 1 0 1431 5 0 1424 20 0 1387

[0230] Epioxytetracycline Conc. μg/ml Female Male 0.05 n.d. n.d. 0.1n.d. n.d. 1 0 1350 5 0 1308 20 0 1343

[0231] Anhydrotetracycline Conc. μg/ml Female Male 0.05 0 1452 0.1 01528 1 0 1614 5 0 1448 20 5 1592

[0232] Anhydrotetracycline Conc. μg/ml Female Male 0.05 0 1381 0.1 01304 1 0 1121 5 0 1269 20 1 1247

[0233] Anhydrotetracycline Conc. μg/ml Female Male 0.05 0 1114 0.1 01120 1 0 1130 5 0 1148 20 0 1128

[0234] Anhydrotetracycline Conc. μg/ml Female Male 0.05 0 1331 0.1 01431 1 0 1309 5 0 1359 20 1 1362

[0235] Anhydrotetracycline Conc. μg/ml Female Male 0.05 0 1582 0.1 01499 1 0 1474 5 0 1619 20 5 1533

[0236] Anhydrotetracycline Conc. μg/ml Female Male 0.05 0 707 0.1 0 14571 0 1437 5 0 773 20 5 1447

[0237] Anhydrotetracycline Conc. μg/ml Female Male 0.05 0 1492 0.1 01426 1 0 1418 5 0 1457 20 8 1499

[0238] Anhydrotetracycline Conc. μg/ml Female Male 0.05 0 1449 0.1 01411 1 0 1397 5 0 1430 20 2 1428

[0239] Anhydrotetracycline Conc. μg/ml Female Male 0.05 0 1339 0.1 01263 1 0 1265 5 0 1284 20 0 1297

[0240] Anhydrotetracycline Conc. μg/ml Female Male 0.05 0 1316 0.1 01358 1 0 1354 5 0 1344 20 1 1312

[0241] Conclusions

[0242] These data show that non-antibiotic analogues of tetracyclineanalogues can be used in place of tetracycline. In the case ofepioxytetracycline, slightly higher concentrations are required torepress gene expression. Neither has parental transmissioncharacteristics substantially different from tetracycline, allowing forthe different effective concentrations.

Example 5

[0243] Effect of Temperature

[0244] All the Preceding experiments were performed at 25° C., thestandard temperature for Drosophila culture. However, the insects in thewild would clearly be exposed to varying temperatures, so weinvestigated the extent to which the efficiency of the system isaffected by temperature As with the recombinant chromosome experiments,40-45 young virgin females and 20-25 young males raised at 25° C. uponfood with the indicated tetracycline supplement were allowed to mate,then transferred to normal (tetracycline-free) food after 3-4 days.These flies were transferred to fresh vials of normal food every day.The numbers of male and female progeny adults in each vial were recordedThese experiments were performed at either 18° C. or 29° C.

[0245] 18° C. Tetracycline Conc. μg/ml Female Male 0.1 8 982 1 10 912 57 871

[0246] Tetracycline Conc. μg/ml Female Male 0.1 6 1065 1 9 1124 5 7 989

[0247] Tetracycline Conc. μg/ml Female Male 0.1 6 695 1 8 816 5 8 785

[0248] Tetracycline Conc. μg/ml Female Male 0.1 2 973 1 9 985 5 5 983

[0249] Tetracycline Conc. μg/ml Female Male 0.1 8 840 1 5 927 5 8 837

[0250] Tetracycline Conc. μg/ml Female Male 0.1 8 832 1 7 879 5 4 818

[0251] Tetracycline Conc. μg/ml Female Male 0.1 6 628 1 3 614 5 5 712

[0252] Tetracycline Conc. μg/ml Female Male 0.1 8 1152 1 12 1122 5 31225

[0253] Tetracycline Conc. μg/ml Female Male 0.1 5 1303 1 14 1218 5 71386

[0254] Tetracycline Conc. μg/ml Female Male 0.1 2 1190 1 4 1213 5 0 1058

[0255] 29° C. Tetracycline Conc. μg/ml Female Male 0.1 0 716 1 0 711 5 0715

[0256] Tetracycline Conc. μg/ml Female Male 0.1 0 781 1 0 749 5 0 741

[0257] Tetracycline Conc. μg/ml Female Male 0.1 0 682 1 0 804 5 0 648

[0258] Tetracycline Conc. μg/ml Female Male 0.1 0 732 1 0 771 5 0 816

[0259] Tetracycline Conc. μg/ml Female Male 0.1 0 749 1 0 737 5 0 718

[0260] Tetracycline Conc. μg/ml Female Male 0.1 0 696 1 0 658 5 0 711

[0261] Tetracycline Conc. μg/ml Female Male 0.1 0 733 1 0 776 5 0 728

[0262] Tetracycline Conc. μg/ml Female Male 0.1 0 765 1 0 702 5 0 773

[0263] Tetracycline Conc. μg/ml Female Male 0.1 0 799 1 0 749 5 0 744

[0264] Tetracycline Conc. μg/ml Female Male 0.1 0 718 1 0 753 5 0 757

[0265] Conclusions

[0266] At low temperature there is a slight leakiness, but only at alevel of <1% escapers. All versions are extremely effective at 29° C.This is important as many of the most important target species forcontrol are tropical and are grown in culture at around 28° C., e.g.Ceratitis capitata, Anopheles gambiae, Aedes aegypti.

Example 6

[0267] The example illustrates material transmission of Tc andTC-repressible lethality using an embryo specific promoter.

[0268] Materials and Methods

[0269] Plasmid Construction

[0270] A bnk promoter fragment of approximately 2 kb was amplified fromplasmid pW⁺2.8 kb bnk rescue fragment (Schejter and Wieschaus, (1993),Cell 75, 373-385) using oligonucleotide primers

[0271] 5′-GCCGAGCTCTTGACGGTTGAAGTACGAATG3′ and

[0272] 5′-CGGCCATTCATATGCGTATATTCACTATG-3′. This fragment was digestedwith and subcloned as a SacI-XhoI fragment into pUHD15-1 (Gossen andBujard, (1992), Proc Natl Acad Sci U S A 89, 5547-51). A XhoI-HpaIfragment containing bnk-tTa was subcloned from this into pW8 (Klemenz etal., (1987), Nucl. Acids Res. 15, 3947-59) digested with XhoI and HpaIto create pP {bnk-tTa}.

[0273] W.T.P-2 (Bello et al., (1998), Development 125, 2193-2202) wasmodified by the addition of two complementary oligonucleotides

[0274] (5′-AATTGCCACCATGGCTCATATGGAAfTCAGATCTG-3′ and

[0275] 5′-GGCCGCAGATCTGATICCATATGAGCCATGGTGGGC-3′) between the EcoRI andNotI sites to provide a consensus translation start sequence (Kozak,(1987), Nucleic Acids Res 15, 8125-48). A cDNA containing the entirecoding region of a Drosophila homologue of Nipp1 (Van Eynde et al.,(1995), J Biol Chem 270, 28068-74) in pNB40 (Brown and Kafatos, (1988),J. Mol. Biol. 203, 425-437) was isolated using the method of(Alphey,(1997), BioTech. 22, 481486) based on a partial sequence obtained by atwo-hybrid screen for Drosophila PPlc-binding proteins (Alphey et al.,(1997), J. Cell Biol. 138, 395-409). The entire coding region and 3′UTRwas cloned between the NdeI and NotI sites of W.T.P-2, modified asabove, to create {pPtRe -Nipp1Dm}.

[0276] Drosophila Culture

[0277] Flies were reared on standard yeast/cornmeal/agar food with ayeast concentration of 45-50 gl⁻¹. Tc-containing food was made to thesame recipe with the addition of tetracycline hydrochloride(Sigma-Aldrich) solution to the appropriate final concentration.

[0278] Histochemistry Embryonic progeny from bnk-tTA/tRe-lacZ crosseswere collected at 12 h intervals and then stained for β-galactosidase asdescribed in Ashburner, (1989), Cold Spring Harbor, NY: Cold SpringHarbor Laboratory Press.

[0279] Results

[0280] Maternal Transmission of Tetracycline

[0281] A mass-reared insect strain homozygous for a dominant lethal geneor genetic system will have no progeny when mated to wild insects. Inthis respect the time of action of the lethal gene is irrelevant.However, for the mass-reared insects to be useful as a control agent weconsider that the time of action of the dominant lethal may be highlyimportant. A lethal phase in adulthood may kill or at least reduce thefitness of the released adults prior to mating. This would clearly becounter-productive. Many agricultural pests damage crops through thefeeding of their larval stages. It would therefore be desirable to killthe progeny as early as possible, preferably as embryos. However,embryos do not feed and so will not take up a dietary repressor(tetracycline) of the lethal genetic system. Insect embryos are alsoimpermeable to most macromolecules, so exogenous tetracycline will notpenetrate. In view of the advantages of an embryonic lethal phase, wetested whether tetracycline ingested by a female Drosophila could passinto her eggs and hence her progeny at sufficient concentration tosuppress the phenotype of a Tc-repressible gene. We used a swain ofDrosophila in which females, but not males, require Tc for viability.Female-specific lethality is due to the expression of a toxic gene(Ras64B^(V12), (Matsuo et al., (1997), Development 124, 2671-80) in thefat body of female larvae and adults (Thomas et al., (2000), Science287, 2474-2476). Growing this strain on food supplemented with 0.1 μg/mlTc is sufficient to suppress expression of the toxic gene, allowing bothmales and females to survive. We reasoned that if Tc ingested by afemale Drosophila could pass into her eggs and hence her progeny, itmight be possible to load the eggs with a high enough concentration toallow survival of the progeny even on media lacking Tc. We found thatallowing parents to feed on food supplemented with Tc at 500 μg/ml orhigher led to the survival of a small proportion of female progeny(Table 1). We tested several other lines and other promoter-killer genecombinations with similar results (data not shown). We concluded that itis possible by feeding a female Tc to introduce enough Tc into herprogeny to repress tTa-dependent gene expression.

[0282] Embryo-specific Expression of tTa

[0283] Of the many genes known to be expressed in Drosophila embryos,the huge majority are also expressed later. For example, the well-knowndevelopmental genes involved in laying down the basic body plan of theembryo are re-used later to pattern the appendages and the imaginaldisks that will form adult structures. For many other embryonic genesthe possibility of later expression has not been rigorouslyinvestigated. bottleneck (bnk) is one of a relatively small number ofgenes reportedly expressed exclusively in embryos. bnk is required foractin filament reorganisation during the cellularisation of theDrosophila embryo between nuclear cycles 13 and 14 (Schejter andWieschaus, (1993), Cell 75, 373-385). Its transcript is present at highlevels only from nuclear cycles 11 to 14. We constructed stabletransformed lines of flies carrying the tTa open reading frame under thecontrol of a bnk promoter fragment. The ability of bnk-tTa to activatetranscription in the embryo and at other developmental stages wasmonitored by using a tta-responsive reporter constructs, tRe-lacZ (Belloet al., (1998), Development 125, 2193-2202). We found that tTa proteinwas expressed in the embryo and that it could direct expression of thereporter construct.

[0284] tTa-dependent transcriptional activation is repressed by Tc. tTabinds to a specific DNA sequence, the tetracycline responsive element(tRe). Tc binds to tTa and this prevents the tTa protein binding to DNA.We therefore attempted to repress reporter gene expression either bysupplementing the parents' food with Tc or by seeding the embryos ontomedia supplemented with Tc. Effective repression of the reporter geneswas achieved by placing the parents on media containing 1 μg/ml Tc forat least two days prior to embryo collection. Seeding embryos onto mediacontaining Tc did not appear to affect reporter gene expression. Thesedata suggest that Tc can enter the egg through the mother duringoogenesis and can affect tTa-mediated transcription at early stages ofdevelopment, but Tc cannot diffuse into embryos from the substrate ontowhich they are laid.

[0285] A “Killer Genen ” Active In Embryos”

[0286] In order to construct a maternal Tc-dependent dominant lethalgenetic 6system, we crossed flies carrying stable insertions of bnk-tTato flies carrying insertions of tRe-Ras64B^(V12) . Our previous studiesbad shown that tRe-Ras64B^(V12) is toxic at later stages in combinationwith a range of female-specific and non-sex-specific tTa lines (Thomaset al., (2000), Science 287, 24742476). To our surprise, embryoscarrying bnk-tTa and tRe-Ras64B^(V12) survived to adulthood irrespectiveof parental or zygotic exposure to Tc (Table 2 and data not shown). Inview of the embryonic reporter gene expression above we concluded thatexpression of Ras64B^(V12) is not toxic, or not sufficiently toxic,during the period when bnk-tTa is active to cause embryonic lethality.

[0287] As ectopic Ras64B^(V12) apparently lacks embryonic toxicity underthe conditions we are using, we placed a different toxic gene under thecontrol of tRe. We chose to use Nipp1Dm, a Drosophila homolog ofmammalian Nipp-1, a nuclear inhibitor of protein phosphatase type 1(1Beullens et al., (1992), J. Biol.Chem. 267, 16538-16544; Van Eynde eal., (1995), J Biol Chem 270, 28068-74). NIPP1 has several advantages asa “Killer gene” in this system. Flies carrying homozygous insertions ofbnk-tTa or tRe-Nipp1 were crossed to each other. Flies fed on mediasupplemented with Tc produced viable F₁ progeny; those on media notsupplemented with Tc did not (Table 2). Furthermore, F₁ survival was notaffected by the presence or absence of Tc in the media on which the F₁were raised. We have therefore constructed an efficient dominant lethalgenetic system repressible by parental dietary Tc. TABLE 1 High doses ofmaternal Tc can suppress tTa in progeny. Tc conc. Day 1 Day 2 Day 3 Day4 Day 5 Day 6 Day 7 Day 8 Day 9 μg/ml ♂ ♀ ♂ ♀ ♂ ♀ ♂ ♀ ♂ ♀ ♂ ♀ ♂ ♀ ♂ ♀ ♂♀ 0.1 93 0 119 0 112 0 141 0 100 0 126 0 89 0 105 0 133 0 1 117 0 135 0122 0 121 0 127 0 101 0 136 0 90 0 119 0 5 112 0 116 0 128 0 111 0 136 0113 0 130 0 119 0 96 0 20 89 0 107 0 107 0 98 0 88 0 102 0 107 0 135 0126 0 100 129 0 136 0 128 0 127 0 135 0 144 0 107 0 96 0 92 0 500 136 288 0 113 0 113 0 87 0 94 0 109 0 141 0 144 0 1000 107 13 140 5 110 0 1410 98 0 129 0 88 0 138 0 105 2000 119 32 102 15 107 12 109 9 109 8 140 2127 0 114 0 123 Tc conc. Day 10 Day 11 Day 12 Total μg/ml ♂ ♀ ♂ ♀ ♂ ♀ ♂♀ 0.1 93 0 131 0 121 0 1363 0 1 94 0 98 0 100 0 1360 0 5 88 0 144 0 91 01384 0 20 143 0 123 0 141 0 1366 0 100 104 0 94 0 115 0 1407 0 500 123 0104 0 124 0 1376 2 1000 124 0 115 0 114 0 1409 18 2000 132 0 115 0 107 01404 78

[0288] A strain homozygous for second chromosome insertions of bothYp3-tTA and tRe-Ras64B^(V12) was tested for the effect of parentaldietary Tc. 40-45 young females and 2025 young males raised at 25° C.upon food with the indicated tetracycline supplement were allowed tomate, then transferred to normal (tetracycline-free) food after 34 days.These flies were transferred to fresh vials of normal food every day for12 days, and then removed on the 13th day. All the vials were incubatedat 25° C. while the progeny developed. The total n umbers of male andfemale progeny emerging as adults were recorded. Survival of femaleprogeny clearly depends on die Tc concentration on which their parentswere raised, and on the length of time between removal of the parentsfrom Tc media and egg laying. TABLE 2 Tc-repressible lethality using anembryo-specific promoter. Tc (μg/ml) Males Females bnk-tTa × tRe-Nipp1Dm 0 0 0 0.1 60 58 1.0 78 82

[0289] Males homozygous for bnk-tta were mated with females homozygousfor either tReRas₆₄B^(V12) or tRe-Nipp1Dm. These flies were raised onmedia lacking Tc, but before mating were placed on food containingvarious concentrations of Tc. They were allowed to lay embryos on thisfood for 9 days, and then the parents were removed. Their adult progenyof each sex were counted. In combination with bnk-tTa, tRe-Nipp1Dm givesTc repressible lethality of both sexes, but tRe-Ras64B^(V12) does not.

Example 7

[0290] Modular Transformation Vector

[0291] This example details the construction of a vector suitable fortransformation to produce an organism containing the lethal geneticsystem of the invention.

[0292] The purpose of this modular vector is to allow the rapid creationof a transformation construct suitable for a given species. In thisexample, the intention is to create a dominant repressible lethal. Thisis achieved by inserting a suitable promoter into this constrict, thenusing it to transform the target species. The promoter is typicallyderived from the target species itself, which is probably the mostdirect and safest way to ensure that the promoter has the desiredspecificity (e.g. female-specific) in the target species. This is not,however, necessary and indeed in the example below we have used amodified actin gene promoter from the silk moth Bombyx morn, with theintention of using it in pink bollworm, a pest of cotton.

[0293] PiggyBac as been used successfully to transform a wide range ofinsects, including Diptera, Coleoptera and Lepidoptera, but it is notnecessarily optimal, nor will Act5C-EGFP be the optimum transformationmarker in every case. The plasmid has been constructed such that thecore elements of the system (tTa, tRe-Nipp1 and insulators) are flankedby unique sites for rare-cutting restriction enzymes (NozI and theSbfI-PmeI-AscI multiple cloning site) to facilitate subcloning theseelements into a new transformation vector. Similarly, alternativeinsulators could be used or an additional insulator inserted 5′ of thenew promoter, to protect against position effects from flankingchromatin.

[0294] The general arrangement of the vector is shown in FIG. 1, andelements are as follows:

[0295] tTa comprises: tTa open reading Same and SV40 polyA signal bothfrom pUHD15-lnco (Gossen and Bujard, 1992) as EcoRI-BamHI. pUHD15-1 wasdigested with XhoI and EcoRI and a oligo pair inserted which destroyedboth these sites and created an AscI site. This plasmid (pUHD5Asc) wasdigested with HpaI and Ba and another oligo pair inserted: tTa 3′linker+5′-gcggccgc ac gggccc a ctcgag cac aagctt c ggtacc ac gaattc-3′ tTa3′linker− 5′-agct gaattc gt ggtacc g aagctt gtg ctcgag a gggccc gtgcggccgc-3′ to create pUHD15Asc3′linker#42.

[0296] tRe-Nipp1Dm comprises: tRe vector W.T.P-2 from Bruno Bello (Belloet al., (1998), Development 125, 2193-2202) modified by insertion ofoligo pair Kozak Spe+/−” between.

[0297] EcoRI and NotI site to give pWTP-KozakSpe. This providesconsensus translation start sequence. Kozak Spe+/−:5′-aattgccaccatggaattcactagtgc-3′ 3′-cggtggtacettaagtgatcacgccgg-5′

[0298] Nipp1Dm cDNA in pNB40 (Brown and Kafatos, (1988), J. Mol. Biol203, 425437) modified to have EcoRI site at start codon, then subclonedas EcoRI-{endfilled NotI} into pWTP-KozakSpe cut with EcoRI and StuI.tRe-Nipp1Dm-hsp70polyA fragment excised as partial XhoI-HindIII andsubcloned into pUHD15 Asc3′linker#43 cut with XboI and HindIII to giveptTatReNipp1#77. The complete predicted sequence of this fragment isappended. NippIDm DNA may be readily prepared by RT-PCR or PCR fromgenomic DNA using this sequence.

[0299] piggyBac and plasmid vector are derived from p3E1.2-white(Handler et al., (1998), Proc Natl Acad Sci U S A 95, 7520-5) from A1Handler. The medfly white gene, originally inserted as a NotI fragmentinto the HpaI site of piggybac, using linkers, was removed by digestionwith NotI and recircularising. A set of extraneous restriction sitesvector sequences (outside piggyBac) was removed by digesting with EcoRIand SalI, end-filling and recircularising, giving p3E1ΔRI-Sal. Thisplasmid was then digested with BgUI and NotI and an oligo pair insertedto add useful restriction sites: piggy linker 2+/−: 5′-ggcc ctcgag agaaggcct gcggccgc tgt ggcgcgcc aga gtttaaac agt cctgcagg-3′ 3′-gagctc tcttccgga cgccggcg aca ccgcgcgg tct caaatttg tca ggacgtcc ctag-5′ theresulting plasmid is pPB-linker2#93.

[0300] The Act5C-EGFP transformation marker was added by subcloning as a4.2 kb XhoI-EcoRV fragment from Act5C-EGFP in pP {CaspeR} (Jean-MarcReichhart) into XhoI-StuI cut pPB-linker2 to give pPB-ActSCEGFP#181.

[0301] The HS4 insulator was added by cutting pJC13-1 (Chung et al.,(1993), Cell 74, 505-14) from Gary Felsenfeld with BamHI andrecircularising, to remove the neo reporter, then excising an HS4 dinner(2×1.2 kb =2.4 kb total) as {endfilled SalI}-KpnI and subcloning intoptTatReNipp1#77 incubated sequentially with HindIII, Klenow DNApolymerae and KpnI (i.e. KpnI cohesive end—endfilled HindIII) to giveptTatReNipp1HS4#101.

[0302] The apoB insulator was added by changing the SpeI site ofapoB3′MAR (Namciu et al., (1998), Mol Cell Biol 18, 2382.91) fromStephanie Namciu to ApaI using the oligo SpeI-ApaI:

[0303] SpeI-ApaI: CTAGAAGGGCCCTT

[0304] The apoB insulator was then subcloned as a 0.8 kb Apal-NotIfragment into ApaI-NotI digested ptTatReNipp1HS4#101.

[0305] An AscI-NotI fragment from ptTatReNipp1HS4#101 was subcloned intopPB-Act5CEGFP#181 to give pRIDL#204

[0306] Examples of Inserting a Promoter:

[0307] 1) A BmA³ promoter fragment of approximately 190bp was amplifiedby PCR from pJP88 (John Peloquin) (Peloquin et al., (2000), Insect MolBiol 9, 323-33) using Platinum Pfx polymerase (Life Technologies) andthe oligos: BmA3 5′: 5′-aaavAATTCTGATAGCGTGCGCGTTAC-3′ BmA3 3′Asc-2: 5′-ggtaggcgcgcc TGGCGACCGGTGGATCCGAATG-3′

[0308] This PCR product was digested with AscI and subcloned intoAscI-PmceI digested pRIDL#204 to give pRIDLI-Bi³

[0309] 2) An Aedes aegypti Vg1 promoter fragment, previously used by usto give femalespecitic expression in the yellow fever mosquito Aedesaegypti, using Platinum Pfx polymerase (Life Technologies) and theoligos: Aedes vg5′ aaac gaattcaccaccaggcagtg Aedes vg3′AscI ggaggcgcgcctcaagtatccggcagctgttc

[0310] This PCR product was digested with AscI and subcloned intoAscI-PmeI digested pRIDL204 to give pRIDL-A.a.Vg1

[0311] For use in plants, the minimal promoter used in combination withthe tetO repeats would be a suitable plant minimal promoter. Forlong-tern stability of expression this would preferably be a minimalpromoter not subject to gene silencing. The promoter driving tTaexpression would suitably be a plant promoter, e.g. the A9 promoter fortapetum-specific expression in a system designed to eliminate pollenproduction in the absence of the repressor.

[0312] The Predicted Sequence of tReNipp1. (XhoI-HindIII)

[0313] nt 1-543 derived from W.T.P.-2 (Bello et al., (1988), Development125:2193,), of which 1-309 contains 7 repeats of the tet operatorsequence (tetO), followed by 98 nt of P element transposase corepromoter, from Camegie 4,-52/+51 relative to transcription start, linkedby a SmaI-PstI linker (synthetic oligonucleotide, GGGCTGCAG) to theleader sequence of bsp70 from CaSpcR-hs (Thummel and Pirrotta, (1991),Dros. Inf. News. 2,) up to the EcoRI site of its polylinker. The nextsection is derived from a synthetic oligonucleotide and provides aconsensus Elation start and some restriction sites, followed by thecoding region of Drosophila Nipp1 and 3′ UTR to polyA sequence from anunpublished cDNA in pNB40 (Brown and Kafatos, (1988), J. Mol. Biol203:425,) up to the NotI site, which has been end-filled and cloned intoan StuI site. The StuI site and subsequent sequence is from W.T.P-2 andis derived from CaSpeR-hs, it is principally trailer (3′ UTR) sequencefrom hsp70flanked by some restriction sites.CTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTAGGAGTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGCTCGGTACGCTTACCGAAGTATACACTTAAATTCAGTGCACGTTTGCTTGTTGAGAGGAAAGGTTGTGTGCGGACGAATTTTTTTTTGAAACATTAACCCTTACGGGCTGCAGTAAAGTGCAAGTTAAAGTGAATCAATTAAAAAGTAACCAGCAACCAAGTAAATCAACTGCAACTACTGAAATCTGCCAAGAAGTAATTATTGAATACAAGAAGAGAACTCTGAATAGGGAATTGGGAATTGCCACCATGGCTCATATGGAATTCATGGCTAACAGCTACGACATACCCAGTTGGGCTGGAAAACCGCCCACTGGCTTACATCTGGATGTGCTAAAGGACGACAAACTAGTACAAAAACTGATGGTGGATGAAAAAAGATGCTATCTATTTGGTCGCAACAGTCAAATGAACGACTTCTGCATAGACCATGCCTCTTGTTCGCGGGTCCACTCGGCGTTTGTCTACCACAAGCACCTCAACATAGCCTACCTCGTGGATCTGGGGTCCACTCATGGCACCTTTATTGGAACACTCAGATTGGAAGCGCACAAGCCCACACAGCTGCAGATTAATAGCACCTTCCACTTTGGGGCTTCTACCCGGAACTACATACTCAGGGAACGACCCTCTGGCCACCACAGCAACATCATGGAAGACCTGCCGCTCAGTGAAACCAGCGATGGCGCTCTCCTGGGCCTGCCCGAAAGCCAAACGGAGCTTGATAATCTTACAGAATACAACACGGCCCACAATCGGCGCATCTCAATGCTGGGCATCGATGATGATACCAATATGCGAAAGCAAAACGCCTTGAAACAGGGACGGCGCACTCGAAATGTCACATTTAACGATGAGGAGATTGTCATCAATCCTGAGGATGTGGATCCTAATGTGGGACGCTTCAGGAACTTGGTACAAACCACTGTGGTGCCCGCCAAGAGGGCTCGCTGCGACGTCAACCATATGGGCATCCATTCGGGCAACAGCAGTTTGTCCAGTGCCAATGCCGCACATGTACACCAAATGTTCCAGCAGAGCCTAGTTGACATGAAGCAGCAGCATAGGGAAATGCCTCCGCCCAATGCGGTGCTCCACTCGCCTACTAATTCCCTATATCAAGGTCTACCGGCCGAAATGCATGGCAAGGGTGACCTAGAGCCCATCTCCCCGCTGAGCATTGGTTCCAAGTTGGGCCTATTGCTCCCGAATCCTGCGCCTGAAGTGTCGCCAGTCTATGACGAAGCTGTGGAGACCTCGACATTGGCTCAAAAGTTGGCCGTCGCTAATGCAAACGTTCGTCGCTTCGGTGAGGATCCGCATGACTCGAGTGGCGAGGGCGATTCGCTGTGCCCACAGAAAAAGAAATACGCCAAGGAAGCATGGCCAGGTCGCAAGCCCATGTTGGGGCAGCTGTAATTGCGTATTAACAAAATAATTAAGATTCCACCTACGATTTTCTCAAGCATATGATTGACAACACACTCTGGAGTAATATTTGTTTATTAGACTTTTAACGTAAAACAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACGAATGCTGCGGCCCCTAATTCCAGCTGAGCGCCGGTCGCTACCATTACCAGTTGGTCTGGTGTCGGGGATCCGTCGACTAAGGCCAAAGAGTCTAATTTTTGTTCATCAATGGGTTATAACATATGGGTTATATTATAAGTTTGTTTTAAGTTTTTGAGACTGATAAGAATGTTTCGATCGAATATTCCATAGAACAACAATAGTATTACCTAATTACCAAGTCTTAATTTAGCAAAAATGTTATTGCTTATAGAAAAAATAAATTATTTATTTGAAATTTAAAGTCAACTTGTCATTTAATGTCTTGTAGACTTTTGAAAGTCTTACGATACATTAGTATCTATATACATGGTTCATTCTACATTCTATATTAGTGATGATTTCTTTAGCTAGTAATACATTTTAATTATATTCGGCTTTGATGATTTTCTGATTTTTTCCGAACGGATTTTCGTAGACCCTTTCGATCTCATAATGGCTCATTTTATTGCGATGGACGGTCAGGAGAGCTCGAATTAACGGGGATCCGTCGACCTGCAGCCCAAGCTT

Example 8

[0314] Models of Insect Control

[0315] Methods

[0316] Thomas et al. (Science 287, 24742476, 2000) presented a simplemathematical model for the effectiveness of insect control programsincluding SIT and various forms of ‘RIDL’ (=‘release of insects carryinga dominant lethal’—used here to indicate the organism and method of thepresent invention), and mentioned that enhanced systems could also beconsidered, including released males being homozygous for dominatefemale lethals (DFLs) at multiple unlinked loci and linking of the DFLto a meiotic drive/segregation distortion system (Thomas et al. 2000,Science 287, 2474-2476). We consider here the impact of these and othersystem enhancements on the effectiveness of insect control.

[0317] In general, we assume that all control programs release aconstant number of males per pest generation (see below). This number(“input”) is given relative to the initial male pest population. Themodel considers discrete generations.

[0318] Females are assumed to select mates proportionately to theirabundance and fitness such that a female will choose a mate of type iwith probability p_(i), such that:

p _(i) =n _(i) r/(S _(j) n _(j) r _(j))

[0319] where n_(i) is the number of male insects of type i and r_(i) isthe fitness of type i relative to wild type males taken to have afitness of 1. The type of insect may depend on its genotype as well asits generation—in particular, we will consider scenarios in whichreleased flies have reduced fitness but their male progeny, regardlessof genotype, have the same fitness as wild type males.

[0320] We consider two scenarios for fertility. In the density-dependentcase, we assume that each female mating with a fertile male produces R₀female offspring—of which the proportion si survive to adulthood wheresi is given by:

S _(i)/(1+(ao _(i))^(b))

[0321] where o_(i) is the number of offspring surviving to the point atwhich density dependence acts (Maynard Smith and Slatkin 1973 Ecology54, 384-391), and a and b are parameters. Rogers and Randolph (1984Insect Sci. Applic. 5, 419-23) consider such a density dependent actswith an SIT program and show that the effectiveness of the SIT proms islargely determined by the natural resilience of the target insectpopulation, characterized by the parameter b (Rogers and Randolph, 1984Insect Sci. Applic. 5, 419-23). An important consideration is the timingof the density dependent mortality, in particular, whether the releasedmales are released before or after the density dependent mechanism actsand whether the RIDL-induced mortality is achieved before or after thedensity-dependent mechanism acts. In the absence of control, such apopulation will remain constant if at its carrying capacity (s_(i)=1/R₀)and will tend to return to that level if perturbed. This would be anappropriate model for an established population. This does notnecessarily mean that no control has been attempted—control methods suchas breeding site elimination will reduce the stable level of thepopulation, rather than the size of the population relative to thestable level.

[0322] In the density-independent case, we assume that each femalemating with a fertile male produces R_(o) female offspring all of whichsurvive to adulthood in the next generation. This is essentially thelimit of the density dependent case—in which the population is so smallthat there is essentially no density-dependent mortality and s_(i)=1.Thus, in the absence of control, if R₀ is greater than 1, the populationwill expand exponentially. This would be an appropriate model for a newintroduction or outbreak of a pest species, or a population recoveringfrom a severe depletion, e.g. due to a short intensive control programmedesigned to reduce the numbers of the target species prior to a RIDL orSIT programme.

[0323] For example, for a very simple system with no density dependence,R₀ equal to 2 and an input of 1.5 sterile males at each generation, themodel would work in the following manner:

[0324] (i) The initial population consists equally of wild type malesand wild type females. All numbers are counted relative to the initialfemale population, so this is 1.0 by definition and the initial wildtype male population is here also 1.0. Since we are only consideringpopulations which normally have equal numbers of males and females, theinitial population of males is always 1.0 in these examples.

[0325] (ii) An input of 1.5 sterile males is made, which is to say 1.5times as many males as there are females in the initial population

[0326] (iii) 60% of females mate with sterile males (since of the 2.5males, 60% are sterile) and produce no offspring. 40% of females matewith wild type males to produce 0.8 female offspring (0.8=R₀ *(0.4*1)females mating with wild type males). They also produce 0.8 maleoffspring.

[0327] (iv) Thus the second generation consists equally of wild typemales and females (0.8 of each).

[0328] (v) An input of 1.5 sterile males is made.

[0329] (vi) 65% of females mate with sterile males (since of the 23males, 65% are sterile) and produce no offspring. 35% of females matewith wild type males to produce 0.56 female offspring (0.56=R₀*(0.35*0.8) females mating with wild type males). They also produce 0.56male offspring.

[0330] and so on until the population is eliminated.

[0331] SIT

[0332] In each case we compare the effectiveness of various versions ofthe RIDL system with that of SIT. For SIT we consider an optimal casewith perfect sex-separation, and 100% sterility. Such SIT is itself ahighly effective control method, and we also consider the level whichthe population would have achieved in the absence of any control, but weshow that various versions of RIDL are much more effective, and thereare many situations in which RIDL can control a pest population whereSIT cannot. These models do not take into account some additionaladvantages of RIDL, for example the greater ease of transporting thesystem to a new species (see vector of Example 7)

[0333] Input

[0334] In general we assume that all control programs release a constantnumber of males per pest generation. This number (“input”) is givenrelative to the initial male pest population. One potential RIDLstrategy involves the release of a mixed-sex population, in theknowledge that one sex will be killed by the lethal effect of asex-specific lethal genetic system at some later point in its lifecycle, e.g. prior to sexual maturity. In the instance where theseindividuals can induce density-dependent mortality in theirconspecifics, (FIGS. 6a and 7 a) we assume that an equal number offemales are released in addition to the males.

[0335] The ability to release at any life cycle stage allows anotherstrategy in which individuals are stored in a dormant state thenreleased simultaneously, allowing a larger release than would otherwisebe the case. For example, embryos of many mosquito species can be storedfor months in relatively dry conditions with little loss of viability,then hatching and larval development induced simply by placing them inan aqueous environment One major advantage of this approach is that amass-re facility could continue to operate during the winter, while thetarget insects are in diapause and hence insensitive to a sterilerelease. In the spring a much larger release could then be used,employing the stored embryos from several generations. Hem, we model theconsequences of storing two generations-worth of factory output andusing this to double the size of the first two release generations (FIG.8). Though the ability to store embryos is not specific to RIDL, an SITprogramme would normally need to grow these embryos up to a laterdevelopmental stage in order to sterilize them by irradiation. Sincefactory rearing space, rather than the availability of embryos, islikely to be the limiting factor, the potential of a RIDL programme torelease at any life cycle stage is critical to this novel strategy. InFIG. 8 we have nonetheless considered the advantage such a releasestrategy would confer on a conventional SIT programme. Each of the plotsin FIG. 8 are based on an earlier plot with a double-sized release inthe first two generations. To compare with conventional SIT without sucha double-sized release, compare these plots with the earlier figuresfrom which they are derived.

[0336] Lethal phase and use of a multi-phase lethal system (MPLS)

[0337] If the requirement is simply to kill all progeny, or all progenyof one sex, then the lethal phase is not important. However, weconsider-that there are several advantages to engineering embryo.specific lethality. The lethal phase must end before the developmentalstage at which the insects are released, or they may lose fitness or dieonce the repressor has been withdrawn, e.g. following release. Embryoniclethality ensures that no larvae emerge to damage crops or animals. Thismay not be important in the case of disease vectors such as mosquitoes,where only the adult stages transmit the disease, but is clearlycritical in the case of many crop pests where it is the voracious larvaethat cause the economic damage. Embryo specific lethality allows thelast and biggest mass-reared generation to be reared on food lacking therepressor, reducing costs and any environmental hazards associated withlarge quantities of Tc. Embryo specific lethality (or other earlylethality) can also.be combined with later sex-specific lethality, e.g.female-specific lethality. We have demonstrated that this allows theconstruction of a stain in which both sex-separation and “sterilization”are automatic consequences of the withdrawal of Tc from the lastgeneration prior to release. We call this system a multi-phase lethalsystem (MPLS), to indicate that there are two different lethal phaseswith different properties. In many rearing/distribution scenarios, thegenetics of such a system appear similar to that of a single-sex releaseof radiation-sterilised males, in that only males are reached and theyhave no viable progeny when mating with wild males in the naturalenvironment. However, there arm two major advantages that are seen inthe models below. Firstly of course the MPLS males have not beenirradiated, and so do not suffer the loss of fitness and longevityconsequent upon irradiation. Secondly, since the requirement is onlythat that the two (or more) lethal phases do not overlap, not that oneof them is specific to embryos, we could arrange that the first lethalphase is after a density-dependent mortality phase in the wildpopulation. For example, in the case of mosquitoes, in order to preventtransmission of most mosquito-borne diseases (e.g. malaria, denguefever, yellow fever) it is only necessary to prevent the females takingheir second blood meal. Killing females as pupae, emerging adults orjust following their first blood meal is would therefore be suitable.The earlier non-sex-specific lethal phase only has to be earlier thanthis, and could therefore be as late as early adulthood, for example.Alternatively, a first lethal phase of late larval/pupal developmentwould be possible. Promoters suitable for all these stages arewell-known—blood-meal inducible genes for killing post-blood meal. etc.Using a lethal phase that first acts later than a density-dependentmortality phase in the wild population means that individuals that willlater die due to the lethal effect of the RIDL system nonethelesscompete for resources with their wild type conspecifics and so tend toincrease the mortality of these wild type conspecifics.

[0338] In the graphs below, SIT and MPLS give the same outcomes, exceptwhere otherwise noted.

[0339] Linking of the DFL to a Meiotic Drive/segregation DistortionSystem

[0340] The meiotic drive system acts to enhance the effectiveness of theRIDL system (FIG. 2). Meiotic drive systems of varying effectiveness areknown in a wide range of species, including Drosophila and mosquitoes.In normal Mendelian inheritance, each of the two homologues of a givenchromosome is equally likely to be inherited, i.e. each has a 50% chanceof being inherited by each individual offspring. The consequence of ameiotic drive/segregation distortion system is that one chromosome ispreferentially inherited. We explored the effect of this by consideringsystems in which the chromosome carrying the RIDL system-ispreferentially inherited by progeny of heterozygotes carrying thischromosome and its homologue from the wild population. Since meioticdrive/segregation distortion systems vary in effectiveness, weconsidered inheritance frequencies of 50% (i.e. no meioticdrive/segregation distortion), 60%, 70%, 80%, 90% and 100%. Higherinheritance frequencies always make the RIDL system more effective.

[0341] Released Males Being Homozygous for DFLs on Multiple Chromosomes

[0342] Unlike SIT, the impact of a RIDL system can potentially beincreased by increasing the copy number of the system within thereleased individuals. We have considered the consequences of releasingmales homozygous for a dominant female-specific lethal at one, two orthru unlinked loci. We found that released males with multiple DFLchromosomes will more effectively control the population size FIG 3).

[0343] Reduced Fitness

[0344]FIGS. 4 and 5 demonstrate the effects of reduced fitness on SITand RIDL systems (with meiotic drive and multiple chromosome systems).Obviously, reduced fitness in released males decreases the effectivenessof these control systems. In FIGS. 4b and 5 b we assume that RIDL maleshave twice the competitive mating fitness of SIT males. This is a veryconservative estimate. Radiation reduces the competitive mating abilityof the irradiated insects (by an estimated two-fold in the case ofmedfly) and also reduces their longevity (by an estimated 2-5 fold inthe case of medfly). This reduces the overall competitive mating abilityby an estimated 4-10 fold in the case of medfly, more in the case of thepink bollworm, less in the case of the screwworm fly. An overalltwo-fold advantage for the non-irradiated RIDL flies over theirirradiated SIT equivalents is therefore a very conservative estimate. Wefound that even this advantage is extremely significant in terms of thecost and effectiveness of a control programme (FIGS. 4b and 5 b).

[0345] Density Dependence Mechanism

[0346] We consider three scenarios:

[0347] (a) density dependent mortality acts before RIDL-inducedmortality and acts on newly released RIDL or SIT insects,

[0348] (b) density dependent mortality acts before RIDL-inducedmortality but does not act on newly released RIDL insects, and

[0349] (c) density dependent mortality acts after RIDL-induced mortalitybut does not act on newly released RIDL insects.

[0350] Scenario (a) models an adult lethal phase and density-dependentmortality acting at the level of adults. An adult lethal phase for RIDLmight be appropriate for malaria vectors, where the females need only bekilled within a week or so after their first blood meal to preventtransmission of the disease. Alternatively, this scenario also models anearlier release and density-dependent stage, which is available forRIDL, where the release population can be released at any life cyclestage, but not for SIT where sex-separation (if used) and irradiationhave to be performed prior to release, restricting the range of lifecycle stages that can be released.

[0351] Scenario (b) is a very important and novel case. This representsa density-dependent mortality that acts before RIDL-induced mortality.In the case of mosquitoes, competition between larvae for resources is alikely stage for density-dependent effects. RIDL-induced mortality cansafely be later than this, as only the adult females transmit disease.Such mortality could be achieved by using a later-acting promoter, suchas that from the vitellogenin gene (for female-specific mortality) inthe vector of Example 7. Tis strategy is also possible using amulti-phase 1lethal system (MPLS) in which the non-sex-specific lethalstage is later than the density dependent mortality. No equivalentstrategy is available for SIT.

[0352] Scenario (c) represents release at a late life cycle stage, e.g.adults, and early RIDL-induced lethality, e.g. as embryos.Density-dependent mortality lies between these. This might representsome crop eating agricultural pests, where the larval stages do thedamage and so it would be inappropriate to release these stages or toarrange for the RIDLinduced mortality to be so late that the larvae havealready done some damage before they die. Unlike scenarios (a) and (b),RIDL has no especial life-cycle-derived advantage over SIT under thisscenario.

[0353]FIGS. 6 and 7 illustrate the benefits of delaying RIDL-inducedmortality until after density dependent mortality, as well as thepotential further benefit of releasing insects (both males and females)prior to the density-dependent mortality. These graphs furtherillustrate the benefits to be gained from increased meiotic drivesystems and multiple chromosome DFL systems. FIGS. 6b, 6 c, 7 b and 7 creveal that SIT can actually lead to a higher stable population thanwould have been the case in the absence of the control programme. Thiseffect has previously been noted by Rogers and Randolph (Rogers andRandolph, 1984 Insect Sci. Applic. 5, 419-23).

[0354] General Points

[0355] For an assumed pattern of productivity and mortality, it is easyto predict that increases in meiotic drive will improve effectiveness,increases in the number of loci with DFLs will improve effectiveness andreduced fitness in released males, relative to wild type males, willdecrease the effectiveness. However, the model demonstrates the relativeimpact of these changes and most importantly demonstrates that reducedfitness may simply act to slow down the control of an insect population,but it can also mean that the insect population cannot be eliminatedwithout a larger input of RIDL or SIT males. Similarly, increasedmeiotic drives can act to improve a given system enough to eliminate aninsect population that would not have been eliminated with a meioticdrive of 50%. Furthermore, not all of the outcomes are intuitivelyobvious. The possibility that SIT can actually lead to a higher stablepopulation than would have been the case in the absence of the controlprogramme has been mentioned above. Additionally, it is clear that undersome circumstances the wild population may actually rise during theearly stages of the control programme and yet still be eradicated in thelonger term. For example, in FIG. 2b with 70% meiotic drive, thepopulation is higher than its initial level from generation 1 togeneration 7, yet is still ultimately controlled.

[0356] Note that in several of the graphs the long dashed line shown inthe key appears in the plot as a continuous or segmented lines. It isnevertheless clear from the context which line is which.

[0357] In detail, the Figures are explained as follows:

[0358]FIG. 2: Meiotic drive system. 50%, 60%, 70%, 80%, 900/o and 100%for a single locus system with no decreased fitness. The bold black lineis the SIT system in each case. The RIDL system is plotted with greylines.

[0359] a R₀ is 1.5 and input is 0.5 (relative to the initialpopulation). SIT maintains the population at a constant level whereasthe RIDL systems quickly reduce the populations. If the population werenot subject to control, by generation 15 we would expect (1.5)15=438times as many insects as the initial population.

[0360] b R₀ is 2.25 and input is 1 (relative to the initial population).The population is not controlled by SIT or RIDL with 50% or 60% meioticdrive. By generation 15 they have 8000, 2200 and 360 times as manyinsects as the initial population, respectively. If the population werenot subject to control, by generation 15 we would expect (2.25)¹⁵=191751times as many insects as the initial population. The populations arequickly brought under control with greater meiotic drive levels.

[0361]FIG. 3: Multiple unlinked loci used in RIDL system The bold blackline is the SIT system in each case. The RIDL system is plotted withgrey lines.

[0362] a R₀ is 1.5 and input is 0.5 (relative to the initialpopulation). SIT maintains the population at a constant level, whereasthe RIDL systems quickly reduce the populations. If the population werenot subject to control, by generation 15 we would expect (1.5)⁼15438times as many insects as the initial population.

[0363] b R₀ is 2.25 and input is 1 (relative to the initial population).The population is not controlled by SIT or RIDL with 1 or 2 loci. Bygeneration 15 they have 8000, 2200 and 34 times as many insects as theinitial population, respectively. If the population were not subject tocontrol, by generation 15 we would expect (2.25)¹⁵=191751 times as manyinsects as the initial population The populations are quickly broughtunder control with a 3 locus RIDL system.

[0364]FIG. 4: Meiotic drive system 50%, 60%, 70%, 80%, 90% and 100% fora single locus system with decreased fitness. The bold black line is theSI system in each case. The RIDL system is plotted with gray lines.

[0365] a R⁰ is 1.5 and input is 0.5 (relative to the initialpopulation). The fitness of SIT and released RIDL insects is 80% of thatof wild type insects. Subsequent generations are assumed to have equalfitness to wild type insects regardless of their parentage. Thepopulation is not controlled by SIT or RIDL with 50% meiotic drive. Bygeneration 15 they have 44 and 1.5 times as many insects as the initialpopulation, respectively. If the population were not subject to control,by generation 15 we would expect (1.5)¹⁵=438 times as many insects asthe initial population. The populations are quickly brought undercontrol with greater meiotic drive levels, despite the reduced fitness.

[0366] b R₀ is 1.5 and input is 1.75 (relative to the initialpopulation). The fitness of SIT is 25% of that of wild type insects,whereas released RIDL insects have 50% of the fitness of wild typeinsects. Subsequent generations are assumed to have equal fitness towild type insects regardless of their parentage. The population is notcontrolled by SIT. By generation 15 it has 23 times as many insects asthe initial population. If the population were not subject to control,by generation 15 we would expect (1.5)¹⁵=438 times as many insects asthe initial population The populations arc quickly brought under controlwith RIDL systems, despite the reduced fitness.

[0367]FIG. 5: Multiple unlinked loci used in RIDL system with decreasedfitness. The bold black line is the SIT system in each case. The RIDLsystem is plotted with grey lines.

[0368] a R₀ is 1.5 and input is 0.5 (relative to the initialpopulation). The fitness of SIT and released RIDL insects is 80% of thatof wild type insects. Subsequent generations are assumed to haveequalitness to wild type insects regardless of their parentage Thepopulation is not controlled by SIT or RIDL with 50 meiotic drive. Bygeneration 15 they have 44 and 1.5 times as many insects as the initialpopulation, respectively. If the population were not subject to control,by generation 15 we would expect (1.5)¹⁵=438 times as many insects asthe initial population. The populations arc quickly brought undercontrol with multiple loci, despite the reduced fitness.

[0369] b R₀ is 1.5 and input is 1.75 (relative to the initialpopulation). The fitness of SIT is 25% of that of wild type insects,whereas released RIDL insects have 50% of the fitness of wild typeinsects Subsequent generations are assumed to have equal fitness to wildtype insects regardless of their parentage. The population is notcontrolled by SIT. By generation 15 it has 23 times as many insects asthe initial population. If the population were not subject to control,by generation 15 we would expect (1.5)¹⁵=438 times as many insects asthe initial population. The populations are quickly brought undercontrol with RIDL systems, despite the reduced fitness.

[0370]FIG. 6: Meiotic drive system. 50%, 60%, 70%, 80%, 90% and 100% fora single locus system with no decreased fitness—in a density-dependentsystem. The bold black line is the SIT system in each case. The RIDLsystem is plotted with grey lines. In all cases a=1, b=2, R₀ is 4.5 andinput is 1 (relative to the initial population).

[0371] a Density dependent mortality acts before RIDL-induced mortalityand acts on newly released RIDL insects. SIT mains the population at aconstant level of 0.8 relative to the initial population, whereas theRIDL systems quickly reduce the populations. If the population were notsubject to control, we would expect the population to remain stable atthe initial size.

[0372] b Density dependent mortality acts before Ridl induced mortalitybut does not act on newly released RIDL insects. The population iseliminated by SIT or RIDL with 50% or 60% meiotic drive. They stabilizethe population at the levels 1.2, 0.4 and 0.3, relative to the initialpopulation, respectively. The populations are quickly brought undercontrol with greater meiotic drive levels. If the population were notsubject to control, we would expect the population to remain stable atthe initial size.

[0373] c Density dependent mortality acts after RIDL-induced mortalitybut does not act on newly released RIDL insects. The population iseliminated by SIT or RIDL with 50%, 60% or 70% meiotic drive. Theystabilize the population at the levels 1.2, 0.75, 0.7 and 0.6, relativeto the initial population, respectively. The populations are quicklybrought under control with greater meiotic drive levels, despite thereduced fitness. If the population were not subject to control, we wouldexpect the population to remain stable at the initial size.

[0374]FIG. 7: Multiple unlinked loci used in RIDL system with nodecreased fitness—in a density-dependent system. The bold black line isthe SIT system in each case. The RIDL system is plotted with grey lines.In all cases a=1, b2, R₀ is 4.5 and input is 1 (relative to the initialpopulation).

[0375] a Density dependent mortality acts before RIDL-induced mortalityand acts on newly released RIDL insects. SIT maintains the population ata constant level of 0.8 relative to the initial population, whereas theRIDL systems reduce the populations. If the population were not subjectto control, we would expect the population to remain stable at theinitial size.

[0376] b Density dependent mortality acts before RIDL-induced mortalitybut does not act on newly released RIDL insects. The population ismaintained at a constant level by SIT or RIDL with 1 locus. Theystabilize the population at the levels 1.2 and 0.4, relative to theinitial population, respectively. The populations are eliminated with 2or 3 locus systems. If the population were not subject to control, wewould expect the population to remain stable at the initial size.

[0377] c Density dependent mortality acts after RIDL-induced mortalitybut does not act on newly released RIDL insects. The population ismaintained at a constant level by SIT or RIDL with 1, 2 or 3 loci. Theystabilize the population at the levels 1.2, 0.75, 0.63 and 0.5, relativeto the initial population, respectively. If the population were notsubject to control, we would expect the population to remain stable atthe initial size.

[0378]FIG. 8

[0379] The plots ad are based on scenario of FIG. 2, with the first tworeleases doubled in size.

[0380] (a) R=1.5, later input=0.5 (b) R=2.25, later input=1

[0381] (c) R₀=1.5, later input=0.5 (d) R₀=2.25, later input=1

[0382] The plots e.g. are based on scenario of FIG. 6, with the firsttwo releases doubled in size.

[0383] (e) see FIG. 6

[0384] (L) see FIG. 6b

[0385] (g) see FIG. 6c

[0386] The plots h-j are based on scenario of FIG. 7, with the first tworeleases doubled in size.

[0387] (h) see FIG. 7a

[0388] (i) see FIG. 7b

[0389] (j) see FIG. 7c

1. A nonhuman multicellular organism carrying a dominant lethal geneticsystem, the lethal effect of which is conditional., wherein the lethaleffect of the lethal system occurs in the natural environment of theorganism.
 2. An organism according to claim 1, wherein the conditionaldominant lethal genetic system is the only recombinant element presentin the organism.
 3. An organism according to claim 1 or 2, wherein theexpression of the lethal genetic system occurs in the absence of asubstance which is absent from the natural environment of the organism.4. An organism according to claim 3, wherein the substance is a dietaryadditive which is not a normal food component for the organism.
 5. Anorganism according to claim 4 wherein the dietary additive is anartificial or synthetic compound, an antibiotic, antibiotic analogue orderivative.
 6. An organism according to any preceding claim, wherein theconditional lethal effect of the lethal system is not dependent upon thetemperature range which occurs in the natural environment of theorganism.
 7. An organism according to any preceding claim wherein thelethal effect of the dominant lethal system is conditionallysuppressible.
 8. An organism according to claim 7, wherein thesuppressible expression system is a repressible by tetracycline or auanalogue or derivative thereof.
 9. An organism according to anypreceding claim, wherein the lethal system is homozygous at more thanone locus.
 10. An organism according to any preceding claim, wherein thelethal system is located on the X chromosome.
 11. An organism accordingto any preceding claim wherein the lethal system has multiple essentialtargets.
 12. An organism according to any preceding claim, wherein thelethal genetic system comprises all or part of the Drosophilia Nipp1Dmgene, or functional equivalent thereof.
 13. An organism according to anypreceding claim wherein the organism is an insect.
 14. An organismaccording to claim 13, wherein the organism is selected from the groupof: Australian sheep blowfly (Lucilia cuprina), Asian tiger mosquito(Aedes albopictus); Japanese beetle (Popilla japonica),White-fringedbeetle (Graphognatus spp.), Citrus blackfly (Aleurocanthus woglumi)Oriental Suit fly (Dacus dorsalis), Olive fruit fly (Dacus oleae),tropical fruit fly (Dacus cucurbitae, Dacus zonatus), Mediterraneanfruit fly (Ceratitis capitata), Natal fruit fly (Ceratitis rosa), Cherryfruit fly (Rhagoletis cerass), Queensland fruit fly (Bactrocera tryoni),Caribbean fruit fly (Anastrepha suspensa), imported fire ants (Solenopisrichieri, Solenopis invicta), Gypsy moth (Lymantria dispar), Codlingmoth (Cydia pomonella), Brown tail moth (Euproctis chrysorrhoea), yellowfever mosquito (Aedes aegypti), malaria mosquitoes (Anopheles gambiae,Anopheles stephansi), New world screwworm (Cochiomyia hominivorar), OldWorld Screwworm (Chrysomya bezziana), Tsetse fly (Glossina spp). Bollweevil (Anthonomous grandis), Damsel fly (Enallagma hageni), Dragonfly(Libellula luctuosa) and rice stem borer (Tryporyza incertulas).
 15. Anorganism according to any of claims 1-12, wherein the organism is aplant which produces pollen.
 16. An organism according to any of claims1, 3-12 and 15, wherein the organism is a plant having a conditionallethal dominant system in combination with one or more transgenes. 17.An organism according to any preceding claim, wherein the lethal geneticsystem is sex-specific or specific to a sexual entity or tissue.
 18. Anorganism according to claim 17, wherein the lethal effect issex-specific or sex-entity specific at one stage of the life cycle ofthe organism, but not specific at another stage.
 19. An organismaccording to claims 17 or 18, wherein the organism does not have adominant sex-specific lethal genetic system which is unconditional andis expressed in every individual.
 20. An organism according to any ofclaims 17-19, wherein the lethal effect is specifc to females or femaletissue.
 21. A method of biological control comprising: i) breeding astock of organisms according to any preceding claim under permissiveconditions; ii) distributing the organisms into the environment at alocus for biological control; and iii) achieving biological controlthrough expression of the lethal system in offspring resulting frominterbreeding of the individuals of the biological control agent withindividuals of the opposite sex of the wild population.
 22. A method ofbiological control according to claim 21, wherein both males and femalesarc distributed.
 23. A method of biological control according to claim21, wherein one sex is distributed.
 24. A method of biological controlaccording to claim 23, wherein there is sex-separation prior to organismdistribution by expression of a sex specific lethal genetic system of anorganism according to claim 17-20.
 25. A method of biological controlaccording to any of claims 21-24, wherein the lethal effect results inkilling of greater than 90% of the target class of the progeny ofmatings between released organisms and the wild population.
 26. A methodfor producing organisms for biological control, comprising the step ofbreeding a stock of or b according to any preceding claim underpermissive conditions.
 27. A method for the sex separation of organisms,wherein the expression of a sex specific dominant conditional lethalsystem according to claim 17 or 18 in an organism is used to kill onesex to leave either. a pure or predominately male or female population;a population in which organisms comprise either male or female tissues;or a population in which organisms are unable to produce functional malegametes or female gametes or both.
 28. A polynucleotide sequenceencoding a conditional dominant lethal genetic system according to anypreceding claim.
 29. A polynucleotide sequence according to claim 28,encoding the Nipp1 gene.
 30. A vector or vectors comprising apolynucleotide sequence according to claim 28 or
 29. 31. A vectoraccording to claim 30, comprising a tetracycline-repressible dominantlethal gene or genetic system.
 32. A vector according to claim 30 or 31,comprising at least one insulator sequence.
 33. A vector according toclaim 32, wherein the insulator sequence is derived from vertebrate DNA.34. A vector according to any of claims 30 to 33, wherein the vectorcomprises modular genetic elements.
 35. A method of conducting a vectorappropriate for imps a dominant lethal genetic system to an organism,comprising the steps of: i providing at least one conditional lethalgenetic system; ii choosing a promoter appropriate for expression of thesystem in the desired organism; iii ligating tic promoter andconditional lethal genetic system, optionally with other components, toproduce a functional vector suitable for transformation; whereintransformation of the vector into the organism produces an organism forbiological control according to the invention.
 36. A cell comprising avector according to any of claims 30-34.
 37. A method for the fieldtesting of transgenic crops, comprising the step of growing a transgenicplant comprising a conditional lethal dominant system under permissiveconditions, and then distributing the plant into the environment whereit is exposed to restrictive conditions.